Anti-hpv-16 e7 antibodies and their use

ABSTRACT

The present invention relates to an anti-HPV-16 E7 antibody obtainable by (a) eliciting an in vivo humoral response against highly purified HPV-16 E7 protein or a fragment thereof in a non-human vertebrate; and (b) affinity-purifying antibodies as obtained in the eliciting-step (a). Furthermore, the invention provides for the use of the anti-HPV-1 E7 antibody/antibodies for the preparation of a diagnostic composition of the (immuno-)histological detection of expressed HPV-16 E7 in a biological sample. Additionally, the invention relates to specific diagnostic compositions as well as to methods producing the same. The invention also provides for kits comprising (an) anti-HPV-16 E7 antibody(ies) of the invention or a diagnostic composition and discloses in vitro methods for the detection of a sexually transmittable disease or cancer, in particular cervical cancer, breast cancer, prostate cancer, anogenital cancer/neoplasia, penile cancer or head and neck cancer by using the described antibodies.

The present invention relates to an anti-HPV-16 E7 antibody obtainableby (a) eliciting an in vivo humoral response against highly purifiedHPV-16 E7 protein or a fragment thereof in a non-human vertebrate; and(b) affinity-purifying antibodies as obtained in the eliciting-step (a).Furthermore, the invention provides for the use of the anti-HPV-16 E7antibody/antibodies for the preparation of a diagnostic composition forthe (immuno-) histological detection of expressed HPV-16 E7 in abiological sample. Additionally, the invention relates to diagnosticcompositions as well as to methods for producing the same. The inventionalso provides for kits comprising (an) anti-HPV-16 E7 antibody(ies) ofthe invention or a diagnostic composition of the invention and disclosesin vitro methods for the detection of a sexually transmittable diseaseor cancer, in particular cervical cancer, breast cancer, prostatecancer, head and neck cancer or anogenital cancer by using the describedantibodies.

Despite an intensive screening program, cervical cancer is one of themost predominant neoplastic diseases in women with a world-wide,incidence second only to breast cancer (Walboomers, 1999). A majoretiological factor in the genesis of cervical carcinoma is the infectionby human papillomaviruses (HPVs), which are small DNA viruses thatinfect epithelial cells of either the skin or mucosa. Of the almost 100different types of HPV that have been characterised to date,approximately two dozen specifically infect genital and oral mucosa(reviewed in zur Hausen, 2000). On the basis of epidemiological andbiochemical data, HPVs are subdivided into two groups. Genital HPVs ofthe high-risk group (most commonly HPV-16, 18) cause cervical cancer andother anogenital cancers while papillomaviruses of the low-risk group(most frequently HPV-6, 11) cause benign genital warts (for review, seeHowley, 1996). PCR based studies have shown that more than 99% ofinvasive cervical cancers world-wide contain high risk HPVs (Walboomers,1999); however, malignant progression occurs only in a small subset ofinfected patients and is typically slow (reviewed in Alexander andPhelps, 2000). Most cervical dysplasia represent squamous cell carcinomaand the main diagnostic tool to detect cervical dysplastic cells isstill based on cytological screening using Pap-smear analysis introducedin 1943 by Papanicolaou (1942) (for review, see Koss, 1989; Meijer andWalboomers, 2000). Although Pap-smear analysis has proven highlyeffective it is difficult to standardise, which is reflected by a highfalse negative error rate of approximately 30% (Walboomers, 1995;Clavel, 1999; Renshaw et al., 2001). Despite the introduction of massscreening programs, the best of which have dropped the mortality ratesby 70%, incidence of cervical cancer in the United States has beenincreasing by about 3% a year since 1986 in spite of an intensificationof the rate of screening (Larsen, 1994).

Since it is medical general knowledge that cervical cancer arises asconsequence of persistent high-risk papillomavirus infections (reviewedin zur Hausen, 2000), the problem could be addressed by the introductionof HPV tests into screening programmes for better identification ofpatients at risk. At present, for clinical applications, PCR and thehybrid capture analysis, both are DNA-detecting methods, are only usefulto a limited extend (reviewed in Milde-Langosch, 2000). Milde-Langoschfurthermore teaches that, besides the molecular biological methods, someantibodies against early HPV-proteins are sold (for example by SantaCruz Biotechnology or Dianova) but these antibodies are, in comparisonto these molecular biological techniques even less sensitive in(immuno)-histochemical analysis.

The major disadvantage of the above discussed molecular biology methodssystems is, that they do only allow to detect viral infection, however,about 5-30% of the normal female population harbours these viruses andonly very few of these develop clinically relevant lesions (von KnebelDoeberitz, 2001). In accordance with this consideration, a high rate oftransient and asymptomatic HPV infections was found especially amongyoung woman (Schiffman and Brinton, 1995). Given the low incidence ofcervical cancer, it may not be useful to apply HPV detection forcervical cancer screening in this age group. Moreover, the PCR basedscreening systems, although highly sensitive, are not widely applied forthe reason that HPV DNA will be detected in a wide range of normalcytological smears resulting in a high rate of false positiveamplification (reviewed in Trofatter, 1997).

It is well established that the expression of E6 and E7, in epithelialstem cells of the mucosa, is required to initiate and maintain cervicalcarcinogenesis (for recent review, see Mantovani and Banks, 2001;Münger, 2001). Thus, a promising way to improve the screening programscould be to measure the expression of the E6 and E7 oncoprotein whichinitiate in a long term process neoplastic transformation in few of theHPV harbouring cells. Since these viral proteins are not expressed innormal cervical squamous epithelia, screening for high risk E7over-expressing cells allows to specifically identify dysplasticlesions. Moreover, progression of pre-neoplastic lesions to invasivecervical cancers is often associated with a continuous enhancedexpression of the E6 and E7 oncoprotein (Schwarz, 1985; Francis, 2000).Similar to these considerations, Klaes (2001) monitored theoverexpression of the cyclin-dependent kinase inhibitor p16 (INK4a), agene which is upregulated in response to E7, as a marker for dysplasticand neoplastic epithelial cells of the cervix uteri. However, p16(INK4A) is only one of several genes which are upregulated in responseto E7 (for review, see McMurray, 2001) and upregulation of p16 (INK4A)expression is not necessary for E7 induced malignant transformation(Giarre, 2001). Furthermore, in view of its central role astumorsuppressor and cell cycle inhibitory protein p16 (INK4A) isupregulated by several other, growth suppressing, stimuli, thus p16(INK4a) is for example well known as target of senescence-inducingpathways (for review, see Bringold and Serrano, 2000). Consequentlyupregulation of p16 (INK4A) might not necessarily reflect the activityof the E7 oncoprotein.

Furthermore, some studies have speculated on the prevalence ofanogenital types of human papillomavirus in prostate cancer and benignprostate hypertrophy. Interestingly, the prevalence of an HPV, inparticular HPV-16 infection in prostate carcinogenesis is highlydisputed. Cuzick (1995, Cancer Sarr. 23, 91-95) review earlier reportson this issue and stress that it is unlikely that common anogenitalpapillomaviruses have an important role in prostate carcinogenesis.Several studies have linked the presence of HPV16 DNA to a risk fordeveloping prostate cancer (e.g. Moyret-Lalle, 1997, Int. J. Cancer 64,125-129; Jerris, 1997 Urology 50, 150-156 or Suzuki, 1996, Prostate 28,318-324), yet, these studies did not provide for a conclusiverelationship between prostate cancerogenesis and HPV-16 activitiesand/or the expression of HPV16 proteins. Wideroff (1996, Prostate 28,117-123) even teaches that HPV infection is not a significant riskfactor for prostate cancer and Anderson (1997, J. Med. Virol. 52, 8-13)confirms the teaching that HPV16 and closely related types are unlikelyinitiations of prostate cancer. Similarly Noda (1998, Urol. Res. 26,165-169) suggests that HPV is not a causal factor for prostatic canceror benign prostatic hyperplasia, and Stickler (1998, Cancer 82,1118-1125 and 1998, Eur. J. Canc. Prev. 7, 305-313) comes to theconclusion that HPV is not associated with prostate carcinomas. Even ifSerth (1998) analysed by single-tube quantitative, competitive PCRsamples from prostate cancers and indicates that in accordance with thisDNA-detection method, HPV16 might contribute to the development of asubset of prostate cancers (Serth (1999), Canc. Res. 59, 823-825),another study of the same year (Saad (1999), Can. J. Urol. 6, 834-838)could not detect HPV DNA in fresh tissue from patients undergoingradical prostatectomy for prostate cancer. Accordingly, the role ofHPVs, in particular HPV16, in prostate cancer remains controversial andelusive.

Several sets of monoclonal antibodies against the HPV-16 E7 oncoproteinor HPV-16 E7 derived peptides have been produced (Sato, 1989; Tindle,1990; Selvey, 1992; Stacey, 1994; Fujikawa, 1994; Zatsepina, 1997) andcommercial preparations are also available (Zymed Laboratories, SanFrancisco, Calif., USA; Santa Cruz Biotechnology, Santa Cruz, Calif.,USA). No antibody, however, was reported as sufficient in sensitivityand specificity to recognise HPV-16 E7 neither in cytological smears norin paraffin embedded sections from biopsies of cervical cancer patients;see Milde-Langosch, 1999. Di Lonardo (2001, Arch Viral 146, 117-125) hasproduced egg yolk antibodies as well as rabbit antibodies against E7oncogenic protein of HPV16. Di Lonardo (2001), loc. cit. stresses thatsome commercial preparations of anti-E7 antibodies are available, butthey suffer severe disadvantages and are not suitable for diagnosticpurposes. Yet, the data provided by Di Lonardo (2001) are not conclusivesince merely the hen antibodies were able to localize HPV-16 E7 in acultured cell line and in a SIL (Squameous Interepithelial lesion)biopsy. However, Di Lonardo (2001) loc. cit. also teaches that therabbit antibodies are not able to detect E7 in immunocytochemistry andstressed that the generated hen antibodies were in a number of casesunable to detect E7 protein in immunostainings of cervical lesions.

Documentation that certain high-risk types of human papillomavirus (HPV)are necessary in the etiology of cervical cancer does it makeconceivable to introduce HPV based tests into screening programmes forbetter identification of patients at risk. Thus it is well establishedthat the expression of the E6 and E7 genes, in epithelial stem cells ofthe mucosa is required to initiate and maintain cervical carcinogenesis.For this reason, a promising way to improve the screening programmescould be the measurement of the expression level of the E6 or E7oncoprotein which initiate, in a long term process, neoplastictransformation in few of the HPV harbouring cells. These viral genes arenot expressed in normal cervical squamous epithelia and the progressionof pre-neoplastic lesions to invasive cervical cancers is presumablyassociated with a continuous enhanced expression of the E6 and E7oncoproteins. For these reasons screening for cells overexpressing highrisk E7 oncoprotein may allow to specifically grade dysplastic lesions.However, expression levels of HPV oncoproteins in cervical carcinoma areunknown so far, due to the lack of specific antisera that wouldrecognize the viral proteins in cervical smears.

Accordingly, there is a need in the art for means and methods whichprovide for reproducible assays for the detection of symptomatic HPVinfections for the detection of the malignant state of a cancerous cellor for the detection of a sexually transmittable disease.

Thus, the technical problem underlying the present invention is tocomply with the needs described above.

In accordance, the present invention provides for an anti-HPV-16 E7antibody obtainable by

a) eliciting an in vivo humoral response against highly purified HPV-16E7 protein or a fragment thereof in a non-human vertebrate; and

b) affinity-purifying antibodies as obtained in the eliciting-step (a).

The term “anti-HPV-16 E7 antibody” as employed herein refers to anantibody, a plurality of antibodies and/or a serum comprising suchantibodies which is/are able to specifically bind to, interact with ordetect the E7 oncoprotein of HPV16 or a fragment thereof. Said term alsorelates to a purified serum, i.e. a purified polyclonal serum. Theantibody molecule is preferably a full immunoglobulin, like an IgG, IgA,IgM, IgD, IgE, IgY (for example in yolk derived antibodies). The term“antibody” as used in this context of this invention also relates to amixture of individual immunoglobulins. Furthermore, it is envisaged thatthe antibody/antibody molecule is a fragment of an antibody, like anF(ab), F(abc), Fv Fab′ or F(ab)₂. Furthermore, the term “antibody” asemployed in the invention also relates to derivatives of the antibodieswhich display the same specificity as the described antibodies. Suchderivatives may, inter alia, comprise chimeric antibodies orsingle-chain constructs. Yet, most preferably, said “anti-HPV-16 E7antibody” relates to a serum, more preferably a polyclonal serum andmost preferably to a purified (polyclonal) serum. The antibody/serum isobtainable, and preferably obtained, by the method described herein andillustrated in the appended examples.

The term “eliciting an in vivo humoral response in a non-humanvertebrate” relates to the provocation of an immune response in anon-human vertebrate, in particular the provocation of an antibodyresponse to HPV-16 E7 or a fragment thereof. Said antibody responsecomprises primary as well as secondary antibody responses to theantigenic challenge with HPV-16 E7 or a fragment thereof. The term“eliciting an in vivo humoral response”, accordingly, relates to theprovocation of an immune reaction involving the production of antibodiesdirected towards the antigen, namely HPV-16 E7 or a fragment thereof.

The term “highly purified HPV-16 E7 protein or a fragment thereof”relates to an isolated HPV-16 E7 protein or fragment thereof, which hasbeen purified to a purity level of at least 95%, more preferably of atleast 96%, even more preferably of at least 97%, particularly preferredof at least 98% and most preferably of at least 99% purity. The purityof HPV-16 E7 protein may be confirmed by methods known in the art,preferably by densitometrical analysis as illustrated in Verdoliva(2000, J. Chormatogr. B. Biomed. Sci. Appl. 279, 233-242),Aboagye-Mathiesen (1992, Prep. Biochem. 22: 105-121) and most preferablyas described in the appended examples. It is preferred that said “highlypurified HPV-16 E7 protein or a fragment thereof” is purified in orderto obtain the corresponding protein or fragment thereof in NMR-grade. Incontext of this invention, the term “highly purified HPV-16 E7 protein”relates to a purified protein E7-preparation which is at least 90%, morepreferably at least 95%, more preferably at least 98% most preferably atleast 99% pure. Accordingly, the highly, purified HPV-16 E7 preparationto be employed in the immunization protocols described herein comprisespreferably less than 5% contaminating, unrelated proteins or proteinfragments. Most preferably, said preparation comprises less than 2%contaminating, unrelated proteins or protein fragments. Purity of thehighly-purified E7 preparation may be measured by methods known in theart which comprise gel stainings (in particular silver stains ofSDS-PAGE followed by densitometric analysis) NMR-measurements or massspectroscopy (MS). The purity of E7 protein or fragments thereof is inaccordance with this invention, most preferably measured by analyzingsamples comprising said E7 or (a) fragment(s) thereof by SDS-PAGE,followed by conventional silver staining and densitometric analysis.Corresponding protocols are detailed in the appended examples. Inaccordance with this invention “highly purified E7 preparations” to beemployed in immunization protocols do not comprise any contaminating,unrelated proteins. According to NMR-analysis the highly-purified E7protein (or immunogenic fragment(s) thereof) is present in a native,partially unfolded structure. Corresponding examples for such apurification is given in the appended examples. In a most preferredembodiment, the highly-purified E7-preparation is a “native, highlypurifed HPV-16 E7 protein” as defined herein below. It is in particularpreferred that said “native, highly purified HPV-16 E7 protein” is afull length protein, comprising preferably 98 amino acids.

The native, highly purified HPV-16 E7 protein or a fragment thereof ispreferably recombinantly produced and, most preferably, said protein orfragment thereof lacks further modifications like additional tags, likeHis-tags or GST-tags.

In accordance with this invention, the term “native, highly purifiedHPV-16 E7 protein” relates to a protein which is correctly folded orrelates to a stretch/fragment of said protein which is correctly foldedand which is soluble, preferably highly soluble. As such, the protein ispurified from E. coli under native conditions and it is not required tounfold/refold the protein by chaotropic agents, such as urea orguanidinium hydrochloride. It is in particular preferred that the nativeHPV-16 E7 protein comprises equivalent amounts of zinc, which isrequired for correct secondary structure of the E7 protein. It is ofnote that the term “native HPV-16 E7 protein” corresponds to the term“native, highly purified HPV-16 E7” in context of this invention andalso comprises naturally occurring variants of HPV-16 E7 protein. Suchvariants are known in the art, as, inter alia, described by Sang Song(1997, Gynecologic Oncology 66, 275-281) or by Ku (2001), Dis. OfColonand Rectum 44, 236-242. The person skilled in the art is easily ina position to determine the folding status of said “native HPV-16 E7protein”, e.g. by CD analysis provided, inter alia, in the appendedexamples. It is envisaged, in accordance with this invention, that anative, highly purified HPV-16 E7 protein (or an immunogenic fragmentthereof) is to be employed in the immunization protocols provided hereinin its native, partially unfolded structure. Therefore, in purified andsoluble form said E7 protein (or its immunogenic fragment) comprises, atleast partially secondary structures like α-helices, β-sheets and turnsand coils. In a most preferred embodiment the E7-protein to be employedin the immunization protocols provided herein comprises 7 to 8%α-helices, 45 to 47% β-sheets, 3 to 5% turns and 40 to 43% coils. Theterms “β-sheet”, “α-helix”, “turn” and “coil” are very well known in theart and, inter alia, described in Brandon/Tooze (1991), “Introduction toProtein Structure”; Garland Publishing Inc., London. The HPV-16 E7fragment to be employed in immunization protocols in accordance withthis invention preferably comprise 6 to 9% α-helices, 43 to 47%β-sheets, 1 to 7% turns and/or 38 to 45% coils. In accordance with thisinvention it was surprisingly found that E7 protein can recombinantly beexpressed and obtained in a soluble, native form as described herein.The use of highly purified recombinant E7 proteins in immunizationprotocols led surprisingly to high quality antibodies specific for saidE7 protein. In contrast to antibodies of the prior art, the antibodiesof the present invention (raised against highly purified, soluble and,preferably, native E7) are capable of specifically detecting E7 inimmunobiological/immunohistochemical samples, like smears. As documentedin the appended examples, prior art antibodies fail to provide forspecific detection means for E7 and, accordingly, a reliable HPVdiagnosis.

The term “fragment of HPV-16 E7 protein” as used herein relates tofragments of a length of at least 40, at least 50, more preferably atleast 60, even more preferably at least 65 amino acid residues of thenative HPV-16 E7 protein. The amino acid sequence of HPV-16 E7 and ofcorresponding variants is known in the art and published in Seedorf(1987, EMBO J. 6, 139-144), Sang Song (1997, loc. cit.) or Ku (2001,loc. cit.). Preferably, said fragment comprises at least the stretch ofamino acids 33 to 98 of HPV-16 E7 as disclosed in Seedorf (loc. cit.).Even more preferably, however, is an E7-protein fragment that comprisesat least amino acids 1 to 70 of HPV-16 E7 as disclosed in Seedorf (loc.cit.).

Preferably, the recombinantly produced HPV-16 E7 protein or its fragmentis expressed in a prokaryotic host, preferably in E. coli. Yet, alsoother expression systems are envisaged which comprise:

Bacterial expression systems, for example, pET System, P_(L) ExpressionSystem, pCAL Vectors, pGEX Vectors, PRO Bacterial Expression System orYeast expression systems, like pESP Vectors, pESC Vectors, PichiaExpression system, YES Vector collection, SpECTRA S. pombe ExpressionSystem, pYD1 System or Insect expression systems, like BacPAK System,Bac-to-Bac Baculovirus Expression System, Bac-N-Blue BaculovirusExpression System, DES: The Drosophila Expression System, InsectselectSystem or Viral expression systems, like AdEasy Adenoviral VectorSystem, AAV Helper-Free System ViraPort Retroviral Gene ExpressionSystem, Adeno-X Expression System, pLXSN System or Mammalian expressionsystems, like PMSG System, pCMV Script, pCI, Creator Gene Cloning &Expression System, Tet-On; Tet-Off Gene Expression System.

As illustrated in the appended example, preferably, said highly purifiedHPV-16 E7 protein or a fragment thereof is purified by a combination ofion exchange chromatography and gel filtration and said purification mayfurther comprise, prior to ion exchange chromatography and gelfiltration, a protein precipitation step.

Ion exchange chromatography is known to the artisan and ion exchangemedia comprise, but are not limited to Mini beads Q, Source 15 Q, Source30 Q, Sepharose High Performance Q, Sepharose Fast Flow Q, Sepharose XLQ, Sepharose Big Beads Q, DEAE, Streamline DEAE (all from AmershamBiosciences, Vienna, Austria), DEAE-cellulose, QA-cellulose,CM-cellulose, SE-cellulose, DE-52 (Whatman, Kent, England) or Agarosebased ion exchangers. Most preferably a Mono QHR 10/10 column (AmershamBiosciences, Vienna, Austria) is employed.

It is of note that also normal gravity flow or FPLC systems may beemployed.

Gel filtration systems and media are also known to the skilled artisanwhich comprise Superdex peptide, Superdex 30, Superdex 200, Superose 6,Superose 12, Sephacryl, Sphadex (all from Amersham Biosciences, Vienna,Austria), Biogel P, Agarose-gel, Fracto-gel or Ultro-gel. A mostpreferred gel filtration system, also employed in the appended examples,is a HiLoad 16/60 Superdex 75 gel filtration column.

Protein precipitation techniques comprise, inter alia, Dextransulphate-, Polyethylene glycol (PEG) 4000-8000-, Acetone-, Protamnesulphate-, Streptomycin sulphate-, pH-shift-precipitations. Preferably,said protein precipitation is carried out by ammonium sulfateprecipitation. More preferably a 30%, most preferably a 38% saturated(NH₄)SO₄-solution is employed.

Accordingly, an example of such a purification method is a three steppurification comprising: 1. protein precipitation, 2. ion exchangechromatography 3. gel filtration. As illustrated in appended example 2,which comprises more details, this precipitation method may preferablybe carried out by an ammonium sulfate precipitation using 30% saturated(NH₄)₂SO₄ solution, most preferably a 38% saturated (NH₄)SO₄-solution,an ion exchange chromatography using a Mono Q HR10/10 column (AmershamBiosciences, Vienna, Austria) and a gel filtration using a HiLoad 16/60Superdex 75 gel filtration column (Amersham Biosciences, Vienna,Austria) It is also envisaged that, as step before the proteinprecipitation or in addition to the protein precipitation, the crudecell lysate is centrifuged, for example at 70 000×g for 1 hour.

As mentioned herein above, the antibodies obtained after eliciting animmune response against the highly purified HPV-16 E7 (preferablynative, highly purified HPV-16 E7) or a fragment thereof are furtherpurified, in particular affinity purified. Preferably, said affinitypurification of the obtained antibodies is carried out over immobilizedHPV-16 E7 protein or a fragment thereof. Most preferably, said HPV-16 E7protein or a fragment thereof is immobilized on PVDF membranes,nitrocellulose, sepharose, agarose, DEAE-cellulose or DEAE. Asillustrated in the appended examples, one possibility of affinitypurifying the HPV-16 E7 or a fragment thereof comprises theimmobilization of HPV-16 E7 or said fragment on PVDF membranes.Immobilized HPV-16 E7 protein is incubated with the polyclonal HPV-16 E7antiserum, washed, and the affinity purified antibodies are eluted by anacid gradient from the immobilized HPV-16 E7 protein. Correspondingprotocols are illustrated in the appended examples.

The elution of bound anti-HPV-16 E7 antibodies may be carried out bymethods known in the art which, inter alia, comprise acid gradients orsalt gradients.

In a particularly preferred embodiment, the HPV-16 E7 protein isprepared as described in Examples 1 and 2, appended hereto.

Most preferably, the “non-human vertebrate” mentioned herein above isselected from the group consisting of rat, mouse, rabbit, chicken,sheep, horse, goat, pig and donkey. Most preferably said vertebrate is achinchilla bastard rabbit or goat.

The antibodies of the present invention provide for the first time areliable tool in the (immuno)-histochemical detection of an HPV-16 E7infection and/or in cancer diagnostic. The antibodies provided hereinare, inter alia, useful in the direct measurement of expressed E7oncoprotein in biological samples, for example in Pap-smears, samplesfrom cervix biopsies, low or high grade squamous, intraepitheliallesions, in samples from prostate biopsies, in particular from fineneedle aspiration biopsies.

It was surprisingly found that the anti-HPV-16 E7 antibodies producedaccording to the above-described method are, in contrast to antibodiesof the prior art, capable of reliably detecting expressed E7 in avariety of biological samples. It is of particular note that theantibodies of the invention are also capable of detecting E7 in fixedmaterial, e.g. in formaldehyde-fixed biological samples. The detectionis also possible in paraffin- or frozen sections of biological samplesand tissue. As documented in the appended examples, the describedantibodies may be employed in (immuno)-histological techniques, likeimmunostainings of biological tissue (e.g. cervix tissue) or in probesderived from fine needle aspiration biopsies (e.g. prostate tissue).

Accordingly, the present invention provides for improved diagnostictools for the detection of an HPV-16 infection. The antibodies of thepresent invention are in particular useful for the detection of HPV-16E7 in Pap-smears.

The detection of, e.g., enhanced E7 oncoprotein expression level by theprovided antibody(ies) allows to identify pre-neoplastic lesions with aparticularly high risk for malignant progression and invasive cancers onhistological probes and/or in cytological smears. This helps to improvecurrent limitations in cancer screening, diagnosis, and therapy control,in particular in cervical and prostate cancer. The described antibodiesprovide for useful tools in the classification of sexually transmitteddiseases or of cancer. Furthermore, these antibodies against highlypurified HPV-16 E7 protein or a fragment thereof recognise the HPV-16 E7oncoprotein in neoplastic cells derived from, e.g., cervical smears, inparaffin- or in frozen-sections from biopsies of patients. Thus, theseantibodies have major diagnostic potential as markers of malignanttransformation in, inter alia, carcinogenesis, e.g. cervicalcarcinogenesis or prostate carcinogenesis.

The antibodies described herein have major advantages over theantibodies of the prior art, e.g. commercially available antibodies as,inter alia, provided by Santa Cruz Biotechnologies or ZymedLaboratories. In contrast to antibodies and antibody-reagents providedby the prior art, the antibody/antibodies/sera described herein arehighly specific and do not provide for high number of “false-positive”signals, i.e. of a “positive” immunobiochemical signal in samples orcells which are HPV-16 negative or which do not express the HPV-16 E7protein or a fragment thereof. Furthermore, the herein describedantibodies are not only highly specific but do also not provide for ahigh number of “false-negative” immunobiochemical signals. Asillustrated in the appended examples the antibodies of the invention maybe, inter alia, tested for this reliability in transfection studies. Forexample, cultured cells, preferably human U2-OS cells may be transfectedwith a vector heterologously expressing E7, for example a vector whichprovides for CMV-driven expression of HPV-16 E7. As a negative control,further U2-OS-cells may be transfected with an expression vector whichdoes not express said E7 protein. “False positive” signals are evaluatedby the amount of cells which are not transfected with the E7-expressingvector, but which, nevertheless, give a positive signal inimmunobiological screenings, e.g. immunofluorescence microscopy.Preferably, less than 15%, more preferably less than 10%, even morepreferably less than 5%, most preferably none of the cultured cellswhich do not (transciently or permanently) express E7-proteins(“negative control cells”) are stained by the antibody described herein.“False negative” signals are evaluated by the amount of cells which arepositively transfected with the E7-expressing vector or which arepositive infected by E7-expressing HPV-16, but which give a negativesignal in immunobiological screenings, e.g. immunofluorescencemicroscopy.

The invention also provides for the use of an anti-HPV-16 E7 antibody ofthe invention for the preparation of a diagnostic composition for the(immuno-)histological detection of expressed HPV-16 E7 in a biologicalsample. Preferably, said (immuno-)histological detection is carried outon Pap-smears (cervical smears), cervical (carcinoma) biopsies orprostate biopsies, like fine needle aspiration biopsies. It is alsoenvisaged that said (immuno)histological detection is carried out onsmears and/or biopsies of anogenital dysplasias. Such dysplasias maylead to, inter alia, anal squamous intraepithelial lesions andneoplasias (ASIL, AIN) or anal, penile and reproductive tract cancers.In this context, also HPV diagnostic, in particular HPV-16 diagnostic isenvisaged which comprises the analysis of samples derived from men,belonging to risk groups of sexually transmittable diseases, likebisexual and homosexual men. Yet, the diagnostic compositions describedherein are useful in diagnostic settings of both, men and women, andindependently from their sexual orientation. Also envisaged is the useof the inventive anti-HPV-16 E7 antibody and the diagnostic compositiondescribed herein in the detection of expressed HPV-16 E7 in smears andbiopsies of head and neck tissue, mamma tissue, prostate tissues, peniletissue, cervix tissue and the like.

Most preferably said diagnostic composition is used for evaluating theacquisition of a sexually transmitted disease or the risk of developingcancer, for measuring the status of an existing sexually transmitteddisease or cancer, or for screening the therapy efficiency in thetreatment of a sexually transmitted disease or cancer.

Furthermore, the invention relates to a method for the preparation of adiagnostic composition comprising the step of formulating the inventiveanti-HPV-16 E7 with a diagnostically acceptable carrier, diluent,buffer, or storage solution. It is also envisaged that in the use or themethod of the present invention, said diagnostic composition furthercomprises suitable means for detection, for example secondary labelledantibodies or fragments thereof.

A variety of techniques are available for labeling biomolecules, arewell known to the person skilled in the art and are considered to bewithin the scope of the present invention. Such techniques are, e.g.,described in Tijssen, “Practice and theory of enzyme immuno assays”,Burden, RH and von Knippenburg (Eds), Volume 15 (1985), “Basic methodsin molecular biology”; Davis L G, Dibmer M D; Battey Elsevier (1990),Mayer et al., (Eds) “Immunochemical methods in cell and molecularbiology” Academic Press, London (1987), or in the series “Methods inEnzymology”, Academic Press, Inc.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,colloidal metals, fluorescent compounds, chemiluminescent compounds, andbioluminescent compounds. Preferred are labels to be detected inimmunohistochemical techniques.

Commonly used labels comprise, inter alia, fluorochromes (likefluorescein, rhodamine, Texas Red, Cy3, Cy5, etc.), enzymes (like,peroxidase, horse radish peroxidase, β-galactosidase, alkalinephosphatase), radioactive isotopes (like ³²P or ¹²⁵I), biotin,digoxygenin, colloidal metals, chemi- or bioluminescent compounds (likedioxetanes, luminol or acridiniums). Labeling procedures, like covalentcoupling of enzymes or biotinyl groups, iodinations, phosphorylations,biotinylations, etc. are well known in the art. It is of note that theantibodies of the invention may also be detected by secondary methods,like indirect immuno-fluorescence. Accordingly, detectably labeledsecondary antibodies may be employed in the methods and uses of thepresent invention.

As mentioned above, direct and indirect detection methods comprise, butare not limited to, fluorescence microscopy, direct and indirectenzymatic reactions and the detection by microscopic means as well asdirect detection by eye-visible signals resulting, inter alia, fromaccumulation of dye-labeled antibodies or the secondary detection ofantibodies. Similarly, as detailed below, the detection of E7 protein bythe inventive antibodies may comprise the detection of soluble orsolubilized E7 protein in fluid samples or solubilized samples. Suchmethods preferably comprise, inter alia, ELISA-, FIA-, CLIA- orRIA-tests (see also below), or the use of test sticks as describedbelow. Commonly used detection assays comprise, accordingly,radioisotopic or non-radioisotopic methods. These comprise, inter alia,Westernblotting, overlay-assays, RIA (Radioimmuno Assay) and IRMA(Immune Radioimmunometric Assay), EIA (Enzyme Immuno Assay), ELISA(Enzyme Linked Immuno Sorbent Assay), FIA (Fluorescent Immuno Assay),CLIA (Chemioluminescent Immune Assay), lateral flow immunoassay, as wellas the use of test sticks detailed herein.

Accordingly, the invention also provides for a diagnostic compositioncomprising the anti-HPV-16 E7 antibody of the invention or obtained bythe method of the invention

Said diagnostic composition may comprise the antibody molecules of thepresent invention, in soluble form/liquid phase but it is also envisagedthat said antibodies are bound to/attached to and/or linked to a solidsupport. Said diagnostic composition may be employed in samples derivedfrom solid tissue as well as in samples which comprise fluid probes.These fluid samples may be selected, inter alia, from blood, serum,plasma, sputum, urine, ejaculate, sperm. It is also envisaged anddescribed herein that solid samples/probes are solubilized be/are theyare tested with the diagnostic composition of the present invention.Yet, in a most preferred embodiment, the antibodies/sera of the presentinvention (and therefore the diagnostic composition) is used on smears,like Pap-smears.

Solid supports may be used in combination with the diagnostic compostionas defined herein or the antibodies, antibody fragments or antibodyderivatives of the present invention may be directly bound to said solidsupports. Such supports are well known in the art and comprise, interalia, commercially available column materials, polystyrene beads, latexbeads, magnetic beads, colloid metal particles, glass and/or siliconchips and surfaces, nitrocellulose strips, membranes, sheets, duracytes,wells and walls of reaction trays, plastic tubes etc. The antibodies ofthe present invention may be bound to many different carriers. Examplesof well-known carriers include glass, polystyrene, polyvinyl chloride,polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses,natural and modified celluloses, polyacrylamides, agaroses, andmagnetite. The nature of the carrier can be either soluble or insolublefor the purposes of the invention. Appropriate labels and methods forlabeling have been identified above. In a preferred embodiment saiddiagnostic composition comprises the use of immobilized inventiveantibodies.

In accordance with this invention, cost-efficient, rapid and reliablediagnostic tests and test kits may be developed. For example, ateststick may be produced that is capable to indicate HPV induced tumordevelopment in cell lysates of cervical smears. Such lysates are oftentaken from material from cervix uteri, which are routinely lysed insample buffers. Yet, the test kits of the invention may also be employedin tests for HPV-16 E7 in other samples, like (blood) serum or lysatesfrom further biopsies or smears, like analogenital biopsies or smears.Such a test, comprising the use of test sticks or other solid matrices,is established on the principle of a ‘lateral flow system’. It is withinthe skill of a person skilled in the art to develop tests/test kits ormeans for testing which comprise, inter alia, the preparation of a teststick directly or indirectly conjugated with the antibodies of theinvention. One, non-limititing example may be the preparation a“cassette housing” with windows for sample application and opticalevaluation of results (comprising test and control lines, respectively)whereby said “housing” comprises a support backing as a carrier for ananalytical membrane, a sample application pad, a conjugate release padand an absorbent pad. The conjugate release pad may be prepared withsubstrates, comprising (conjugated) anti-E7 antibody of this invention,whereby said conjugation may, inter alia, be gold- or latex conjugation.The analytical membrane area in the test window may, inter alia, beprepared with different reagents in separated lines fixed to saidmembrane. It is envisaged that the testline carries the inventiveanti-E7 antibody and the control lines may comprise E7 protein as wellas (an) secondary antibody antibodies, like (an) anti-rabbit (oranti-goat or the like) antibody or antibodies. Furthermore, a/thecontrol line may comprise other detections means for further/othersample compounds. In the test illustrated here, the function of thecontrol lines is to monitor the efficiency of the test/teststick and theconjugated antibodies and to exclude false positive and negative resultsby interfering substances. Similar assays and test means are known inthe art and comprise, inter alia, pregnancy tests based on specificantibody-antigen interactions. The test stick described herein may notonly be employed in cell lysates of tissue(s) to be tested but also inbody fluids, like blood, serum, plasma, sputum, urine, ejaculate, spermand the like.

In a further embodiment the invention provides for a kit comprising ananti-HPV-16 E7 antibody of the invention or a diagnostic composition ofthe invention.

Advantageously, the kit of the present invention further comprises,optionally (a) buffer(s), storage solutions and/or remaining reagents ormaterials required for the conduct of medical, scientific or diagnosticassays and purposes. Furthermore, parts of the kit of the invention canbe packaged individually in vials or bottles or in combination incontainers or multicontainer units. The kit may also comprise aninstruction sheet to carry out the (diagnostic) methods of the presentinvention.

The kit of the present invention may be advantageously used, inter alia,for carrying out the (diagnostic) methods of the invention and could beemployed in a variety of applications referred herein, e.g., asdiagnostic kits, as research tools or medical tools. Additionally, thekit of the invention may contain means for detection suitable forscientific, medical and/or diagnostic purposes, like e.g. secondaryantibodies as described above. The manufacture of the kits followspreferably standard procedures which are known to the person skilled inthe art.

In another aspect the present invention relates to an in vitro methodfor the detection of a sexually transmittable disease or cancercomprising the steps of

(a) incubating a biological sample with anti-HPV-16 E7 antibodies of theinvention; and

(b) measuring and/or detecting specifically-bound anti-HPV-16 E7antibodies whereby the presence, the absence or the amount ofspecifically-bound anti-HPV-16 E7 antibodies is indicative for saidsexually transmittable disease or cancer. It is further preferred thatthis in vitro method comprises a further step (c), whereby in said step(c) the presence, the absence or the amount of specifically-boundanti-HPV-16 E7 antibodies of step (b) is compared to the presence, theabsence or the amount of specifically-bound anti-HPV-16 E7 antibodies ina negative or a positive control sample or in both control samples.

The measurement and/or detection of specifically “bound anti-HPV-16 E7antibodies” may be carried out as described above, for example by thedetection of directly or indirectly labelled, bound antibody moleculesof the invention. Said measuring and detection methods may also compriseautomated and/or computer-controlled detection methods.

Such in vitro methods of the invention are also illustrative in theappended examples and may be, inter alia, employed to detect thepresence or absence of an HPV16 infection, to evaluate whether a HPV16infection is merely transient or an asymptomatic HPV16 infection. It isof note that the antibodies of the present invention may be employed inthe above described method in order to evaluate the absence or presenceof a proliferative disorder, like, e.g. cervix carcinoma, prostatacarcinoma, breast cancer, anogenital cancer, penile cancer and head andneck cancer. Furthermore, the antibodies may be employed to evaluate theclass of a proliferative disorder, for example it can be evaluatedwhether a prostatic carcinoma is HPV16 dependent or independent. It is,e.g., envisaged that patients whose serum comprises prostatic-specificantigen (PSA) or who have a positive result in fine-needle aspirationbiopsies of prostatic tissue are further examined for the presence orabsence of HPV-16 E7, employing the antibodies of the invention andmethods disclosed herein. Such a diagnostic method allows for thedistinction of HPV16-positive and HPV16-negative prostate carcinomas andthe medical intervention may be chosen accordingly.

As mentioned above, the biological sample is preferably a cervix or aprostatic sample, most preferably a Pap-smear or a fine needleaspiration biopsy.

In contrast to previous technology, namely the detection of HPV-16 DNAin prostate cancer biopsies, the detection of high-level HPV-16 E7expression in prostate cancer samples allows the conclusion that inthese samples the E7 oncoprotein, which is the major transformingprotein of the virus, is actively expressed. The person skilled in theart knows that expression of the E7 oncoprotein in any cell results inthe inactivation of several important tumor suppressor mechanisms, asreviewed in Zwerschke (2000, Adv. Cancer Res. 78, 1-29). This indicatesthat there is a high risk for malignant progression of this lesion. Ithas to be stressed that currently physicians do not considerHPV-dependent malignant progression of prostate cancers, since detectionof HPV oncoproteins in prostate cancer specimens was not possible withthe techniques of the prior art. The antibodies described herein allowsignificant progress in clinical research. Furthermore, it isanticipated that with the advent of specific antiviral drugs and/ortreatments directed against high-risk papillomaviruses, the detection ofHPV-16 E7 in prostate cancer specimens will direct the physician to newmodes of treatment for this important malignancy.

In accordance with this invention, the biological sample to be testedand/or evaluated with the inventive anti-HPV-16 E7 antibody may be asolid sample as well as a soluble/solubilized sample. Even if one of themost preferred uses of the inventive antibody/antibodies comprises thediagnostic use in immunohistochemical assays, in particular on smears,further methods of diagnosis employing the inventive antibodies areenvisaged in this invention. These further methods are described andillustrated herein and comprise the use of solid and non-solid phaseimmunoassays, like ELISA-, RIA-tests or the use of (antibody-covered)tests sticks, magnetic or polystyrol beads and the like.

Besides samples derived from cervix, anogenital tissue head- and necktissue and/or prostatic tissue, it is also envisaged that the inventiveanti-HPV-16 E7 antibody is employed in diagnostic samples derived frommamma/mamma tissue. The antibody of the present invention isparticularly useful in screening of mamma tissue obtained from patientswho suffer or had suffered from a cervix carcinoma and may develop, e.g.due to metastasis, a mamma carcinoma. Accordingly, the present inventionalso relates to an in vitro method for detection of a mamma/breastcancer, in particular of a mamma cancer in a patient who suffers or whohas suffered from, in particular a cervix carcinoma/cervical cancer.Said in vitro method comprises the incubation of mamma tissue (solid orsolubilized) with anti-HPV-16 E7 antibodies of the invention and themeasurement and/or detection of specifically-bound anti-HPV-16 E7antibodies, whereby the presence, the absence or the amount ofspecifically bound anti-HPV-16 E7 antibody is indicative formamma/breast cancer. In particular, a positive signal of specificallybound E7 antibody of the present invention is indicative for a mammacarcinoma/breast cancer, in particular a mamma carcinoma being asecondary tumor or a metastasis from a primary tumor, like a cervixcarcinoma or an anogenital cancer.

The invention, accordingly, provides for the use of an anti-HPV-16 E7antibody, a diagnostic composition or a kit of the invention in an invitro method for the detection of a sexually transmittable disease orcancer. Said sexually transmitted disease is, preferably anHPV16-infection or said cancer is cervical cancer, breast cancer,prostate cancer, anogenital cancer/anogenital neoplasia (AIN), penilcancer or head and neck cancer. The feasibility of a successfulHPV-diagnostic, in particular HPV-16 E7 diagnostic on smears isdescribed in the appended examples.

In another embodiment, the present invention provides for a method forproduction of an anti-HPV-16 E7 antibody comprising the steps of

(a) eliciting an in vivo humoral response against highly purified,HPV-16 E7 protein or a fragment thereof in a non-human vertebrate; and

(b) affinity-purifying antibodies as obtained in the eliciting-step (a).

In a most preferred embodiment, the highly purified HPV-16 E7 proteinsor a fragment thereof to be used in the immunization protocol describedherein and illustrated in the appended examples is a native, highlypurified HPV-16 E7 protein or a fragment thereof. The term “native” asused in accordance with this invention is explained herein above andillustrated in the appended examples. With respect to the preferredembodiments the same applies, mutatis mutandis, as described hereinabove for the anti-HPV-16 E7 antibody.

The Figures show:

FIG. 1: Purification of the HPV-16 E7 oncoprotein. Bacterial expressedrecombinant HPV-16 E7 was stepwise purified by ammonium sulfateprecipitation, anion-exchange chromatography on MonoQ and gelfiltrationon a Sephadex G75 column. (A, B) Samples were separated by gelelectrophoresis, and purification was documented by coomassie stainingof the fractions as indicated. Purity of the HPV-16 E7 protein wasconfirmed by Western blotting using a monoclonal anti E7 antibody (SantaCruz, Vienna, Austria) (C).

FIG. 2: Test of the affinity purified anti-HPV-16 E7 antibodies (14/3)in westernblot analysis. A. Purified GST and GST-HPV-16 E7 proteins wereseparated by SDS-polyacrylamide gel electrophoresis, and the GST-HPV-16E7 protein was detected by westernblotting. B. The HPV-16 E7 expressingcells E7/2 and the control cells were subjected to lysis. Subsequentlylysates were separated by SDS-polyacrylamide gel electrophoresis andprobed with antibodies to HPV-16 E7 and beta actin (input control), asindicated.

FIG. 3: Detection of HPV-16 E7 after transient expression in humancells. U-2OS cells were transiently transfected with expression vectorsfor HPV-16 E7, as indicated. At 26 h post transfection, cells wereprocessed for indirect immunofluorescence microscopy and viewed by usinga confocal scanning system. Cells were stained with anti-E7 antibodiesclone 14/3 (α-HPV-16 E7), preimmune serum (control), TroPro3 (nucleus)or both anti-E7 antibodies and TroPro3 (α-HPV-16 E7/nucleus), asindicated.

FIG. 4: Immunoperoxidase staining of paraffin sections of normal cervixand cervical carcinomas with affinity purified polyclonal antibodiesagainst HPV-16 E7. Paraffin sections of normal cervix and a cervicalcarcinoma were immunostained for HPV-16 E7 by the immunoperoxidasemethod as described in material and methods. (A) In cervical carcinomatissue, epithelial cells are negative with the preimmunserum. (B, C, E,G) In cervical carcinoma tissues anti-HPV-16 E7 antibodies stainvirtually all cells in the tumor islets. (D) In normal cervical tissue,epithelial cells are negative with these antibodies. (F) In cervicalcarcinoma tissues staining by the anti-HPV-16 E7 antibodies can becompeted out by preincubation of the antibodies with purified HPV-16 E7antigen. (H) Control, no staining of cervical carcinoma tissues wasobtained by adding only the horse radish conjugated secondary antirabbit IgG.

FIG. 5: Immunoperoxidase staining of cells obtained from prostatecarcinoma patients. Biopsies were taken from 60 prostate carcinomapatients and samples from 60 patients were applied to an object slidetogether with negative controls. These slides are known to the expert as“tissue microarrays” (Skacel, 2002, Appl. Immunohistochem. Mol. Morphol.10, 1-6). Tissue micorarrays were stained with antibodies to HPV-16 E7as described in FIG. 4 for cervical biopsies. In this experiment, asubset of the carcinoma biopsies stained positive for HPV-16 E7, whereasother biopsies from different prostate cancer patients stained negative.

FIG. 6: Cells from surface layers of the ectocervical epithelium werespread out on glass object slides and immunoperoxidase stained by theanti-HPV-16 E7 antibodies (brown). The cells were counterstained withHemalaun (grey/blue) and viewed by brightfield microscopy. The HPV-DNAstatus of the specimens was analyzed by PCR. (A) No brown staining wasobserved in cells from normal (Pap II) HPV-DNA negative ectocervicalsmear. (B) Cells from HPV-16 DNA positive cytological abnormal (PapIIID) ectocervical smear were stained brown by the antibodies.

FIG. 7: Expression of the HPV-16 E7 oncoprotein in biopsies derived fromcervical carcinoma patients. Three HPV-16 positive cervical carcinomasand seven HPV-16 negative cervical tissues were analysed for theexpression of the HPV-16 E7 protein. Lysates, 0.5 mg each, wereseparated by SDS-polyacrylamide gel electrophoresis, and the HPV-16 E7protein was detected by Western blotting. As controls, lysates fromCaSki cells, an established cervical carcinoma cell line (obtained fromDKFZ Heidelberg, Germany), NIH3T3 mouse fibroblasts and NIH3T3/16E7cells, a cell line derived from NIH3T3 cells by stable transfection withthe pMOHPV16E/ expression vector (Edmonds (1989), J. Virol. 63,2650-2656 ), were assayed.

FIG. 8: Three different preparations (see appended example 1) ofrecombinantly expressed HPV 16 E7 protein were evaluated to detect, thepurity of the preparations and the reproducibility of the appliedmethods. The amount of proteins separated per lane was 0,1 μg HPV16-E7protein. The gel was silver stained according to Heukeshoven and Dernick(in R. Westermeier et al. 1990; ISBN 3-527-28172-X) for 30 min. The gelwas scanned using a Fluor-S™ Multi-Imager system (BIORAD). Crosssections of defined lanes were saved as TIFF images using Quantity One(Quantitation Software by BIORAD). Evaluation of the gel-bands wasperformed by using TOTALLab evaluation software Version 1.1. The sum ofall pixels over the entire length of one lane was assumed to beequivalent to 100% of protein applied (0.1 μg/lane). E7 concentrationand purity was 98.0% (A), 98.3% (B) and 98.2% (C).

FIG. 9: A preparation of recombinantly expressed and highly purifiedHPV-16 E7 protein as described herein was evaluated for secondarystructure elements in the native folded protein in a physiologicalsolvent by CD spectroscopy. The native protein to be employed forimmunization protocols is folded into secondary structure elements likeβ-sheets (45-47%), coils (40-43%), α-helices (7-8%) and turns (3-5%).Yet, also fragments of the native, highly purified E7 proteins asdescribed herein may also be employed in immunization protocols.

FIG. 10: Comparison of immunohistochemical staining of HPV-16 E7 proteinin paraffin embedded cervical carcinoma tissue sections (consecutivesections from one tissue slice) by two different monoclonal anti HPV-16E7 antibodies (Santa Cruz, Zymed) and the polyclonal anti HPV-16 E7antibody described herein. Immunohistochemical staining was performed asdescribed in Example 8. Antibodies was diluted according tomanufacturers protocol. (A) In cervical carcinoma tissues anti HPV-16 E7antibodies described herein stain virtually all cells in the tumorislets. (B,C) No clear signal was obtainable in cervical carcinomatissues by the monoclonal anti HPV-16 E7 antibodies ED17 (Santa Cruz)and 8C9X (Zymed). In the latter cases, a high and apparently unspecificbackground is not restricted to the area that is cytologicallyrecognized as tumor tissue, but were also present in the non-tumortissue.

FIG. 11: Comparison of indirect immunofluorescence detection of HPV-16E7 protein in transiently transfected U-2OS cells by the polyclonal antiHPV-16 E7 antibodies described herein and two commercialized monoclonalanti HPV-16 E7 antibodies (Santa Cruz, Zymed). The staining wasperformed as indicated in FIG. 3 and Example 7.

The invention is illustrated by the following examples:

EXAMPLE 1 Construction of the Bacterial Expression Vector for HPV-16 E7

The HPV-16 E7 oncogene was amplified from the vector pX-HPV-16 E7(Mannhardt et al., 2000) by PCR using Pfu DNA polymerase as EcoRIrepair/BamHI fragment. The sequence was inserted into the bacterialexpression vector pET3a (Studier and Moffaft, 1986) prepared as NdeIrepair/BamHI fragment generating the bacterial HPV-16 E7 expressionvector pET3a-HPV-16 E7/clone 17. The sequence encoding for HPV-16 E7 wasverified by sequencing.

EXAMPLE 2 Expression and Purification of Recombinant HPV-16 E7 Protein

The expression vector pET3a-HPV-16 E7/clone 17 was transformed into E.coli strain BL21 (DE3) pLysS. The bacteria were grown to OD₆₀₀=0.5 andthe expression of the E7 protein was induced for 3 hours at 37C.° byadding IPTG (Biomol, Hamburg, Germany) to a final concentration of 0.4mM. Bacteria were harvested by centrifugation for 10 minutes at 5 000×g.The cell pellets were frozen in 20 ml ice-cold lysis buffer (50 mM KCl,20 mM H₂KPO₄ [pH 7.8], 50 mM DTT, 5% glycerol, 1μg/ml leupeptin, 1 mMPMSF, 1 mM NaF and 10 μg/ml Aprotinin) per liter bacterial culture.Cells were thawed on ice and lysed by sonication with glass beads(Sigma, Vienna, Austria) using a Sonifier 250 (Branson, Geneva,Switzerland). After centrifugation at 70 000×g for 1 hour, thesupernatant was ammonium sulfate precipitated using 30% saturated(NH₄)₂SO4 solution. The (NH₄)₂SO4 pellet was dissolved in 4 ml MonoQloading buffer (150 mM Tris/Cl pH 7.8, 10 mM NaCl, 10 mM DTT and 5%glycerol) and dialysed against MonoQ loading buffer. The dialysed probewas centrifuged at 10 000×g for 10 minutes and loaded onto a MonoQHR10/10 column (Amersham Biosciences, Vienna, Austria) via a 10 mlSuperLoop (Amersham Biosciences, Vienna, Austria). Using a linear saltgradient the E7 protein eluted from the anion-exchange-column at 600 mMNaCl. 15 fractions were collected. The E7 containing fractions werepooled and loaded onto a HiLoad 16/60 Superdex 75 gel filtration column(Amersham Biosciences, Vienna, Austria) and run with the gelfiltrationbuffer (150 mM Tris/Cl pH 7.8, 150 mM NaCl and 10 mM DTT). At a flow of0.5 ml/min. Gels were stained with Coomassie brilliant blue and thestained gel was evaluated by scanning, using the Adobe PhotoshopSoftware and a Micro Tec Scan Maker 8700 Image Scanning Device. Thisgenerated a profile of relative optical density which was used todetermined the integral corresponding to the E7 peak. With this softwareit is possible to calculate the percentage of total OD units which arerepresented by the E7 peak. As judged by densitometrical analysis the E7fractions resulting from this run were more than 98% pure (e.g. analyzedby silver-stained SDS-PAGE), or more than >99.5% pure (e.g. judged byCoomassie-stained SDS-PAGE) and were concentrated using a Centriprep10ultrafiltration filter (Amicon, Vienna, Austria). The identity of theE7-protein was confirmed by Western Blot (FIG. 1B) and through a peptidemass fingerprint (PMF) (FIG. 1C).

A further protocol for the purification of E7-protein comprises thefollowing steps:

The expression vector pET3a-HPV-16 E7/clone 17 was transformed into E.coli strain BL21 (DE3) pLysS and preserved as glycerol stock. LB- orNZCYM-medium (25 ml) containing 100 μg/ml Ampicilline (Biomol, Hamburg,Germany;) and 25 μg/ml Chloramphenicol (Sigma, Vienna; Austria;) wasinoculated with the glycerol stock and grown over night at 37° C. to afinal OD₆₀₀ of 1.5. The next day NZCYB medium, containing 100 μg/mlAmpicillin and 25 μg/ml Chloramphenicol and 2 ml Glucose/I, wasinoculated with 1% of the over night culture and grown at 37° C. to anOD₆₀₀ of 0.4. Culture volume was 400 ml per 2000 ml aeration flask. AtOD₆₀₀=0.4 E7 expression was induced by adding IPTG (Biomol, Hamburg,Germany) to a final concentration of 0.4 mM. Two hours after inductionbacteria were harvested by centrifugation for 10 minutes at 5 000×g. Thedrained cell pellets were either stored at −80° C. until further use (upto 3 month) or redissolved in ice-cold lysis buffer (50 mM KCl, 20 mMH₂KPO₄ [pH 7.8], 50 mM DTT, 5% glycerol, 1-μg/ml leupeptin, 1 mM PMSF, 1mM NaF and 10 μg/ml Aprotinin) at a ratio of 2 ml fresh lysis buffer perpellet derived from 100 ml bacterial culture. When the pellet had beenstored at −80° C. lysis buffer was added directly to the frozen materialand cells were thawed on ice. For the following purification proceduretwo pellets from 400 ml E. coli culture each were used. Pellets wereredissolved by repeated pipetting and lysed by sonication with glassbeads (Sigma, Vienna, Austria) using a Sonifier 250 (Branson, Geneva,Switzerland) on ice. The sonified lysate was centrifuged at 70 000×g for1 hour and the supernatant stored on ice. The remaining pellet wasredissolved in lysis buffer (again 2 ml fresh lysis buffer per pelletderived from 100 ml bacterial culture) and sonified and centrifugated asstated above. Supernatants were pooled, cooled on ice and subjected to atwo-step ammonium sulphate precipitation procedure.

To prepare a saturated Ammonium sulphate solution 75 g of (NH₄)₂SO₄ wereadded to 100 ml of 50 mM. NaCl, 150 mM Tris/HCl pH 7.8. Ammoniumsulphate was dissolved at RT and the saturated solution was cooled downon ice. (The cooled solution contained a few crystals of precipitated(NH₄)₂SO₄ indicating 100% saturation.) The saturated (NH₄)₂SO₄ solutionwas prepared freshly prior to use.

The lysate was made 15% ammonium sulphate by adding 15 parts of cold,saturated (NH₄)₂SO₄ solution to 85 parts of cold lysate (5.65 mlsaturated (NH₄)₂SO₄ to 32 ml lysate). The mixture was stirred gently onice for 30 min and centrifuged at 4° C. for 30 min at 30 000×g. Thesupernatant (i.e. 15% (NH₄)₂SO₄) was removed and made 38% ammoniumsulphate by adding 1 part of cold, saturated (NH₄)₂SO₄ solution to 2.7parts of cold supernatant (14 ml saturated (NH₄)₂SO₄ to 37.65 mlsupernatant). The mixture was stirred gently on ice for 30 min andcentrifuged at 4° C. for 30 min at 30 000×g.

After discharging the supernatant, the pellet was dissolved in dialysisbuffer (150 mM Tris/HCl pH 7.8, 10 mM NaCl, 10 mM DTT and 5% glycerol)at a ratio of 1 ml per pellet derived from 100 ml bacterial culture.Dialysis was performed at 4° C. for 12 hours applying 3 buffer changes.Dialysis tubings with a molecular weight cut-off of 10 000 dalton wereused; the total volume of dialysis buffer was 250 times the samplevolume. DTT was added prior to every buffer change. (For 8 ml ofdissolved (NH₄)₂SO₄-pellet, 3×670 ml dialysis buffer were used). Thedialysed probe was centrifuged at 10 000×g for 10 min and thesupernatant loaded (flow=1 ml/min) onto a MonoQ HR 10/10 anion-exchangecolumn (Amersham Biosciences, Vienna, Austria) equilibrated to 10% MonoQbuffer B (MonoQ buffer A: 150 mM Tris/HCl pH 7.8, 10 mM DTT (added priorto use) and 5% glycerol; MonoQ buffer B: 150 mM Tris/HCl pH 7.8, 1MNaCl, 10 mM DTT added prior to use) and 5% glycerol;). The MonoQ columnwas washed with 2 column volumes (CV) (flow=4 ml /min) 10% buffer B, andeluted in multi step gradient at a flow rate of 2 ml/min: 10% -47% B (2CV), 47% B (2CV), 47% -100% B (2 CV). At 47% buffer B (470 mM NaCl) E7eluted in a prominent peak over 3 fractions of 1 ml each. E7 containingfractions were individually loaded onto a HiLoad 16/60 Superdex 75 gelfiltration column (Amersham Bioscienees, Vienna, Austria) and eluted ata flow rate of 0.5 ml/min with the gelfiltration buffer (150 mM Tris/HClpH 7.8,150 mM NaCl and 10 mM DTT (added prior to use)); fraction volumewas 2 ml. E7 containing fractions from 3 runs were controlled onSDS-PAGE followed by coomassie stain. E7 fractions of highest puritywere pooled and the protein concentration was determined according toBradford. The pool was diluted with gelfiltration buffer to a finalconcentration of 1 mg/ml and frozen in aliquots for further use. Thetotal yield from 800 ml E. coli culture was approximately 14 mg ofnative, highly purified HPV-E7 in NMR-grade.

A further protocol for the purification of E7-protein comprises thefollowing steps:

The expression vector pET3a-HPV-16 F-E/clone 17 was transformed into E.coli strain BL21 (DE3) pLysS and preserved as glycerol stock. LB- orNZCYM-medium (25 ml) containing 100 μg/ml Ampicilline (Biomol, Hamburg,Germany;) and 25 μg/ml Chloramphenicol (Sigma, Vienna; Austria;) wasinoculated with the glycerol stock and grown over night at 37° C. to afinal OD₆₀₀ of 1.5. The next day, 99 parts of NZCYB medium, containing100 μg/ml Ampicillin and 25 μg/ml Chloramphenicol and 2 ml Glucose/I,was inoculated with 1 part of the over night culture and grown at 37° C.to an OD₆₀₀ of 0.4. Culture volume was 400 ml per 2000 ml aerationflask. At OD₆₀₀=0.4 E7 expression was induced by adding IPTG (Biomol,Hamburg, Germany) to a final concentration of 0.4 mM. Two hours afterinduction bacteria were harvested by centrifugation for 10 minutes at 5000×g. The drained cell pellets were either stored at −80° C. untilfurther use (up to 3 month) or redissolved in ice-cold lysis buffer (50mM KCl, 20 mM H₂KPO₄ [pH 7.8], 50 mM DTT, 5% glycerol, 1-μg/mlleupeptin, 1 mM PMSF, 1 mM NaF and 10 μg/ml Aprotinin) at a ratio of 2ml fresh lysis buffer per pellet derived from 100 ml bacterial culture.When the pellet had been stored at −80° C. lysis buffer was addeddirectly to the frozen material and cells were thawed on ice. For thefollowing purification procedure two pellets from 400 ml E. coli cultureeach were used. Pellets were redissolved by repeated pipetting and lysedby sonication with glass beads (Sigma, Vienna, Austria) using a Sonifier250 (Branson, Geneva, Switzerland) on ice. The sonified lysate wascentrifuged at 70 000×g for 1 hour and the supernatant stored on ice.The remaining pellet was redissolved in lysis buffer (again 2 ml freshlysis buffer per pellet derived from 100 ml bacterial culture) andsonified and centrifugated as stated above. Supernatants were pooled,cooled on ice and subjected to an ammonium sulphate precipitationprocedure.

The lysate was made 38% ammonium sulphate by adding 38 parts of cold,saturated (NH₄)₂SO₄ solution to 62 parts of cold lysate (19.6 mlsaturated (NH₄)₂SO₄ to 32 ml lysate). The mixture was stirred gently onice for 30 min and centrifuged at 4° C. for 30 min at 30 000×g. Aftercarefully discharging the supernatant, the pellet was dissolved indialysis buffer (150 mM Tris/HCl pH 7.8, 10 mM NaCl, 10 mM DTT and 5%glycerol) at a ratio of 1 ml per pellet derived from 100 ml bacterialculture.

Dialysis was performed at 4° C. for 12 hours applying 3 buffer changes.Dialysis tubings with a molecular weight cut-off of 10 000 dalton wereused; the total volume of dialysis buffer was 250 times the samplevolume. DTT was added prior to every buffer change. (For 8 ml ofdissolved (NH₄)₂SO₄-pellet, 3×670 ml dialysis buffer were used). Thedialysed probe was centrifuged at 10 000×g for 10 min and thesupernatant loaded (flow=1 ml/min) onto a MonoQ HR 10/10 anion-exchangecolumn (Amersham Biosciences, Vienna, Austria) equilibrated to 10% MonoQbuffer B (MonoQ buffer A: 150 mM Tris/HCl pH 7.8, 10 mM DTT (added priorto use) and 5% glycerol; MonoQ buffer B: 150 mM Tris/HCl pH 7.8, 1MNaCl, 10 mM DTT added prior to use) and 5% glycerol;). The MonoQ columnwas washed with 2 column volumes (CV) (flow=4 ml/min) 10% buffer B, andeluted in multi step gradient at a flow rate of 2 ml/min: 10% -47% B (4CV), 47%B (2CV), 47%-100% B (2 CV). At 47% buffer B (470 mM NaCl) E7eluted in a prominent double peak over 4 fractions of 1 ml each. E7containing fractions were individually loaded onto a HiLoad 16/60Superdex 75 gel filtration column (Amersham Bioscienees, Vienna,Austria) and eluted at a flow rate of 0.5 ml/min with the gelfiltrationbuffer (150 mM Tris/HCl pH 7.8, 150 mM NaCl and 10 mM DTT (added priorto use)); fraction volume was 2 ml. E7 containing fractions from 4 runswere controlled on SDS-PAGE followed by coomassie stain. E7 fractions ofhighest purity were pooled and the protein concentration was determinedaccording to Bradford. The pool was diluted with gelfiltration bufferto, a final concentration of 1 mg/ml and frozen in aliquots for furtheruse. The total yield from 800 ml E. coli culture was approximately 14 mgof native, highly purified HPV-E7 in NMR-grade.

Three different preparations of recombinantly expressed HPV 16 E7protein were evaluated to document, the purity of the preparations andthe reproducibility of the applied methods. Material generated on Aug.8^(th,) 2002 (used to immunise chinchilla rabbits; preparation “A”), and2 production lots from Dec. 17^(th,) 2002 (lot 1 used to immunise goats;lot 2 used to prepare an E7-affinity column; preparations “B” and “C”)were run on a 12.5% SDS-PAGE under reducing conditions (2.5%β-Mercaptoethanol). The amount of proteins separated per lane was 0.1 μgHPV16-E7, determined according to Bradford, using BSA as standard. Thegel was silver stained according to Heukeshoven and Dernick (in R.Westermeier et al. 1990; ISBN 3-527-28172-X) for 30 min. The gel wasscanned using a Fluor-S™ Multi-Imager system (BIORAD). Cross sections ofdefined lanes were saved as TIFF images using Quantity One (QuantitationSoftware by BIORAD). Evaluation of the gel-bands was performed by usingTOTALLab evaluation software Version 1.1.

FIG. 8 shows the results from densitometric evaluation of threeindependent preparations A, B and C. Results were calculated fromseparation gels. A light background-staining in the stacking gel,derived from the sample buffer, was observed. Since the light backgroundstaining on top of the separation gel was found in every lane, it isassumed to be derived from an irrelevant compound from the samplebuffer. Prior to evaluation, the background was subtracted from eachlane separately. The sum of all pixels over the entire length of onelane was assumed to be equivalent to 100% of protein applied (0.1μg/lane). Peaks were evaluated be recalculating the pixel-intensity ofevery protein band found into % of the total protein amount per lane. E7concentration was 98.0% (A), 98.3% (B) and 98.2% (C). The curves shownin FIG. 8 are original traces from scanned lanes exported as MS-Excelfiles as the used set-up did not allow to print evaluated curvesdirectly.

Circular Dichroism spectroscopy (CD) Measurements of HPV-16 E7 Proteinin Solution

Circular Dichroism (CD) is observed when optically active matter absorbsleft and right hand circular polarized light slightly differently. CDspectra for distinct types of secondary structure present in peptides,proteins and nucleic acids are different. The analysis of CD spectra cantherefore yield valuable information about secondary structure ofbiological macromolecules. In our case Circular Dichroism Spectroscopyis used to gain information about the secondary structure of nativeproteins and polypeptides in solution. The CD is a function ofwavelength and is measured with the CD spectropolarimeter JASCO J-715.(See Circular Dichroism and Optical Rotary Dispersion of Proteins andPolypeptides, A. J. Alder, N. J. Greenfield and G. D. Fasman, Meth.Enzymology 27, 675 (1973)).

A preparation of recombinantly expressed and highly purified HPV-16 E7protein as described herein was further evaluated for secondarystructure elements that will occur in the native folded protein in aphysiological solvent. Because of 7 Cysteins in the E7 molecule, the E7protein tends to build di- and multimeres with proteins in vicinity bydisulfide bridges. Patrick et al, 1992, JBC 265 (10):6910, describes,that no such disulfide-bonds exist inside the native E7 molecule. It wasdemonstrated that 3 cysteins are accessible to solvent, while cysteinsin the two concerved Cys-X-X-Cys motifs are likely involved to be partof a zinc-finger motif. For this reason inclusion of a reducingsubstance in solvent like DTT (or 2-ME) results in monomeric native E7protein particles (DTT and 2-ME do not have any denaturing effect). Inaddition, the amount of DTT in CD measurement is diluted to the lowestconcentration that might be possible to adhere reducing conditions.Repeated measurements was carried out in 8 μl of a 50-100 μmolar proteinsolution in NMR buffer (20 mM H₂KPO₄, 50 mM KCl, 10 mM NaCl, 10 mM DTT,pH 7.5) diluted in in 80 μl of a. dest.

The obtained measurement data were interpreted by the calculationprogram of the CD spectropolarimeter JASCO H-715 (H-700 SecondaryStructure Estimation for Windows, version 1.10.02, Jasco). TheCD-spectrum and structural data are shown in FIG. 9. The HPV-16 E7protein is folded into secondary structure elements like β-sheets(45-47%), coils (40-43%), α-helices (7-8%) and turns (3-5%).

EXAMPLE 3 Generation of Polyclonal HPV-16 E7 Antibodies:

Purified preparations of the HPV-16 E7 protein were used to producehighly specific polyclonal anti-HPV-16 E7 antibodies in chinchillabastard rabbits (Charles River, Germany). 1^(st) injection: 700 μlcomplete Freund's adjuvant (Sigma, Vienna, Austria) was mixed with 500μg HPV-16 E7 protein dissolved in 700 μl PBS by sonication (Bransonsonifier 250, level 5-7, 3×10 seconds). A total of 300 μg HPV-16 E7protein was injected. 1^(st) boost: 32 days after the first injection,500 μl incomplete Freund's adjuvant was mixed with 500 μg 16 E7 proteindissolved in 500 μl PBS by sonication. A total of 500 μg HPV-16 E7protein was injected. 2^(nd) boost: 28 days after the first boost, 500μl incomplete Freund's adjuvant was mixed with 500 μg 16E7 proteindissolved in 500 μl PBS by sonication and a total of 500 μg HPV-16 E7protein was injected. 3^(rd) boost: 27 days after the second boost, 500μl incomplete Freund's adjuvant was mixed with 500 μg 16 E7 proteindissolved in 500 μl PBS by sonication. A total of 500 μg HPV-16 E7protein was injected. Bleeding was done 10 days after the third boost.In particular, small aliquots of sera were tested in western blot 10days after the first, second and third boost (second, third and forthinjection). A first and clear signal was obtained after the third boost.day application of HPV16 E7 bleedings −3 pre-immune serum taken 1immunisation with 500 μg 16 E7 incomplete FA 33 1^(st) boost with 500 μg16 E7 in incomplete FA 43 1^(st) test bleeding 61 2^(nd) boost with 500μg 16 E7 in incomplete FA 71 2^(nd) test bleeding 88 3^(rd) boost with500 μg 16 E7 in incomplete FA 98 final bleeding

A good immune response was also achieved by using 150 μg and 300 μgHPV16 E7 as antigen respectively. The immunisation schedule was asstated above.

A further protocol for the generation of a polyclonal HPV-16 E7 antibodycomprises the generation of said antibody/serum in goat. Said generationwas carried out as follows:

Highly purified preparations of the HPV-16 E7 protein (see example 2)were used to produce highly specific polyclonal anti-HPV-16 E7antibodies in goats. 1^(st) injection: 1100 μl complete Freund'sadjuvant (Sigma, Vienna, Austria) was mixed with 1000 μg HPV-16 E7protein dissolved in 1000 μl G75 gel filtration buffer (example 2:150 mMTris/HCl pH 7.8, 150 mM NaCl and 10 mM DTT) according to a“syringe-method” (Oxf. Univ. Press; 2000: Practical approach series;ISBN 0-19-963711-3 Vol. Immunoassays; Edited by J. P. Gosling; p.28). Atotal of 1000 μg HPV-16 E7 protein was injected. 1^(st) boost: 28 daysafter the first injection, 1100 μl complete Freund's adjuvant was mixedwith 1000 μg 16 E7 protein dissolved in 1000 μl G75 gel filtrationbuffer by the syringe-method. A total of 1000 μg HPV-16 E7 protein wasinjected. 2^(nd) boost: 28 days after the first boost, 1000 μlincomplete Freund's adjuvant was mixed with 1000 μg 16E7 proteindissolved in 1000 μl G75 gel filtration buffer by the syringe-method. Atotal of 1000 μg HPV-16 E7 protein was injected. 3^(rd) boost: 28 daysafter the second boost, 1000 μl G75 gel filtration buffer by thesyringe-method. A total of 1000 μg HPV-16 E7 protein was injected. Smallaliquots of sera were tested in western blot 10 days after the first,second and third boost (second, third and forth injection). Finalbleeding was done 14 days after the third boost. A first and clearsignal was obtained after the third boost. day application of HPV16 E7bleedings −3 pre-immune serum taken 1 immunisation with 1000 μg 16 E7incomplete FA 29 1^(st) boost with 1000 μg 16 E7 incomplete FA 39 1^(st)test bleeding 57 2^(nd) boost with 1000 μg 16 E7 in incomplete FA 672^(nd) test bleeding 85 3^(rd) boost with 1000 μg 16 E7 in incomplete FA95 3^(rd) test bleeding 99 final bleeding

EXAMPLE 4 AffinityPurification of Polyclonal HPV-16 E7 Antibodies

Glutathione-S-transferase (GST-HPV-16 E7) and GST (control) wereexpressed from the expression vectors pGEX4T-GST-HPV-16 E7 and pGEX4T(Mannhardt, 2000; Mol Cell Biol 20:6483-95) in the E. coli strain DH5α.Expression was induced by adding IPTG to a final concentration of 1 mMto a 200 ml bacterial culture at OD₆₀₀=1.0. The bacteria were washedonce in PBS and lysed in PBSDT (1.5 mM KH₂PO₄, 8.1 mM Na₂HPO₄ [pH 7.4],2.7 mM KCl, 137 mM NaCl, 0.2 mM phenylmethylsulfonyl fluoride [PMSF], 1mM NaF, 1 mM dithiothreitol [DTT] and 0.5% Triton X-100) by sonicationusing a Branson sonifier 250. The lysates were centrifuged at 4 000×gfor 10 minutes and afterwards at 30 000×g for 30 minutes to remove thecell debris. The recombinant proteins were purified byaffinity-chromatography using the glutathione sepharose 4B system(Amersham, Vienna, Austria). Clear supernatants were incubated for 3hours at 4° C. with 150 μl glutathione sepharose 4B beads, which wereprior, equilibrated in cold (4° C.) PBSDT. After the binding intervalthe beads were washed 4 times in 5 ml of PBSDT and stored at 4° C. inPBSDT. Purity of the preparation was controlled by western blottingusing an anti E7 antibody (clone ED17, Santa Cruz, Vienna, Austria) andby Coomassie staining. Aliquots of 200 μg of bound GST proteins wereseparated on a 12.5% SDS-PAGE and transferred to a polyvinylidenedifluoride (PVDF) membrane (NEN, Boston, USA) by electro blotting. PVDFmembranes were dried and the proteins were crosslinked to the membraneby UV irradiation. The protein bands were stained with Ponceau Ssolution, excised from the PVDF membrane, destained and transferred tomicrofuge tubes.

Subsequently, the fragments were incubated with the polyclonal rabbitHPV-16 E7 antiserum for 2 hours at room temperature and washed 3 timesin PBS-T (1.5 mM KH₂PO₄, 8.1 mM Na₂HPO₄ [pH 7.4], 2.7 mM KCl, 137 mMNaCl and 0.5% (vol/vol) Tween 20). Each fragment was then eluted bythree 30 seconds washes with 5 mM glycine-HCl, [pH 2.3], 500 mM NaCl,0.5% (vol/vol) Tween 20, 10 μg/ml BSA in volumes of 500 μl; theseeluates were immediately neutralized by the addition of Na₂PO₄ to afinal concentration of 50 mM. The purified antibodies were concentrated4 times using a centriprep YM-3 centrifugal filter (MilliporeCorporation, Bedford, USA). After the pH 2.3 elution, fragments werefurther washed with three similar aliquots of PBS-T and 10 μg/ml of BSA,followed by three washes with 3 M NH₄SCN, 150 mM KCl, 10 mM NaPO₄ [pH6.0], and 10 mg/ml BSA. The procedure was repeated 5 times

A further protocol for affinity purification of polyclonal HPV-16 E7antibodies comprises the following:

Three different columns were used to purify polyclonal HPV-16 E7antibodies from animal serum by affinity chromatography.

Column 1: (column to purify total IgG from antiserum). A HiTrap ProteinG HP column (Amersham Biosciences, Vienna, Austria) was used accordingto the manufacturers protocol to isolate total IgG from antiserum.

Column 2: (pre-column without antigen to adsorb unspecific antibodies tothe affinity matrix): 2.8 g of freeze dried CNBr-activated sepharose 4B(Amersham Bioscieces, Vienna, Austria) were activated according to themanufacturers protocol and transferred into coupling buffer (100 mMNaHCO₃, 500 mM NaCl, pH 8.3) containing 13 mg of NIH 3T3 fibroblastscell lysate (determined according to Bradford). Coupling was performedfor 2 hours at room temperature in a 50 ml Falcon tube attached to arotating platform. Once coupling was completed, the affinity matrix waspacked into a XK16 FPLC column (Amersham Biosciences, Vienna, Austria)by gravity. The settled gel-bed (10 ml) was then washed with 5 columnvolumes of coupling buffer and 5 column volumes of blocking buffer (1Methanolamine, pH 8.0). The column was then left at room temperature for2 hours with out agitation to block remaining active groups, andthereafter washed with 5 column volumes high pH buffer (100 mM Tris/HCl,500 mM NaCl, pH 8.0) and 5 column volumes low pH buffer (100 mMNa-acetate, 500 mM NaCl, pH 4.0). The cycle high pH-wash/low pH-wash wasrepeated 5 times. Finally the column was attached to the FPLC system andequilibrated to running buffer (PBS, 200 mM NaCl, 5 mM EDTA, 0.05% NaN3,pH 7.4). Protein contents (Bradford) of coupling buffer before and aftercoupling, and of all through-runs and wash buffers collected, revealed acoupling efficiency of approximtatly 80% of NIH-3T3 proteins to thecolumn. The ligand density was 1 mg/ml gel-bed; the column volume was 10ml.

Column 3: (affinity column, carrying purified HPV16-E7 protein toisolate polyclonal HPV16-E7 antibodies). As affinity matrixCNBr-activated Sepharose 4B (Amersham Biosciences, Vienna, Austria) wasused. Preparation of the column was as stated above (column 2), but withrecombinant, purified HPV16-E7 (examples 2A-2C) used as ligand. Prior tocoupling, the ligand was dialysed (from 150 mM Tris/HCl, 150 mM NaCl, 10mM DTT, pH 7.8) into coupling buffer as Tris would interfere with thecoupling procedure. The optimal ligand density for affinity purificationwas found to be 1 mg antigen per ml gel-bed. For the experimentdescribed below, an affinity column of a bed-volume of 3 ml, carrying1.5 mg of HPV16-E7 was used.

Purification of the Polyclonal HPV16-E7 Antibody:

Antiserum was diluted 1+9 in running buffer (PBS, 200 mM NaCl, 5 mMEDTA, 0.05% NaN₃, pH 7.4). Diluted material was filtered trough a 0.45μm sterile filter and passed over a 1 ml Protein G column using an AktaPrime system (Amersham Biosciences) at a flow rate of 1 ml/min. Thecolumn was extensively washed with running buffer until the baseline waszero (5 ml/min). Total IgG was eluted in 1 ml fractions (1 ml/min) with100 mM Glycine, 0.05% NaN₃, pH 2.5 into 1.5 ml reaction vials containing50 μl of 3 M KH₂PO₄/K₂HPO₄-buffer pH 7.4 to neutralize the low pH of theelution buffer.

IgG containing fraction (4×1.050 ml) were pooled, topped up to 10 mlwith running buffer and loaded onto column 2 equilibrated with 10 columnvolumes of running buffer. Material was passed over the pre-column at aflow rate of 5 ml/min for 60 min in a closed circle to remove antibodiesthat would bind unspecifically to the CNBr activated sepharose matrix,and to immobilised proteins other than HPV16-E7. Thereafter the adsorbedmaterial was collected (still 10 ml) and pooled with 5 column volumes ofrunning buffer used to wash loosely bound, but probably specificantibodies from the pre-column. Finally the material (15 ml) was passedover the affinity column (column 3, equilibrated in running buffer) at aflow rate of 5 ml/min in a closed circle until an equilibrium wasreached. The column was then washed (5 ml/min) with PBS, 1M NaCl, 5 mMEDTA, 0.05% NaN₃, pH 7.4 until the baseline reached zero. Afterre-equilibration into running buffer (10 column volumes), polyclonalanti HPV16-E7 antibodies were eluted (1 ml/min) with 100 mM Glycine,0.05% NaN₃, pH 2.5 into 1.5 ml reaction vials containing 50 μl of 3 MKH₂PO4/K₂HPO₄ buffer to neutralize the low pH of the elution buffer(fraction size was 1 ml). After elution, the column pH was set back toneutral by passing 10 column volumes of 1M Tris/HCl pH 7.4, 10 columnvolumes of 3 M KSCN, 150 mM KCl, 10 mM KH₂PO₄/K₂HPO₄, pH 7.4 and 20column volumes of running buffer through the system.

Anti HPV16-E7 antibody containing fractions were pooled and testedfurther like stated below. It was found that the pre-column (column 2,carrying NIH-3T3 cell lysate as ligand) in some cases could be omitted.Pooled eluates from column 1 (protein G column) were diluted 1+9 inrunning buffer and directly applied to column 3 (affinity column).

EXAMPLE 5 Test of the Affinity Purified HPV-16 E7 Antibodies

The affinity purified HPV-16 E7 antibodies specifically recognize HPV-16E7 in cell lysates from HPV-16 E7 expressing mammalian cells in westernblot experiments (FIG. 1). HPV-16 E7 was also detectable in human U-2-OScells transiently transfected with a HPV-16 E7 expression vector byindirect immunofluorescence microscopy using the confocal scanningsystem (FIG. 2). Furthermore, the antibodies recognize HPV-16 E7 inimmunohistochemical experiments done in paraffin-embedded sections ofcervical carcinomas derived from biopsies of HPV-16 positive patients(FIG. 3). Biopsies from 12 carcinoma patients were analyzed and positivesignals were obtained with the E7 antibody in all 12 cases. Furthermore,in two cases of cervix biopsies which had previously been classified byPCR-methods as “HPV-16 negative”, the antibody described herein was ableto specifically detect expressed E7.

EXAMPLE 6 Western Blot (Immunoblot) Analysis

Cell extracts were separated on a 12.5% sodium dodecyl sulfate(SDS)-polyacrylamide gel, and proteins were transferred to a PVDFmembrane (NEN, Boston, USA). The membrane was incubated in blockingbuffer (0.05% Tween 20/5% low fat milk powder in PBS) for 1 hour at roomtemperature, washed in blocking buffer and incubated with the firstantibody (affinity purified polyclonal rabbit anti-HPV-16 E7 antibody)for 1 hour at room temperature. After washing in blocking buffer,PBS/0.05% Tween 20 and PBS/5% low fat milk powder the membrane wasincubated with the second antibody (peroxidase-conjugated anti-rabbitIgG, Promega, Mannheim, Germany) for 45 minutes at room temperature. Themembrane was washed, and the bound antibodies were visualized by usingthe chemiluminescence Western blotting detection system (NEN, Boston,USA).

EXAMPLE 7 Indirect Immunofluorescence Analysis

U-2-OS cells were cultured in DMEM+10% FCS. For transient expression ofcDNAs, cells were grown to about 80% confluence on glass coverslipscoated with 0.05% gelatin. Transfection of the expression vectorpJ4HPV-16 E7 (Massimi, et al., (1997) J. Gen. Virol. 78, 2607-2613) wasperformed by using Effectene (Qiagen, Hilden, Germany). 24 hpost-transfection, cells were prepared for indirect immunofluorescenceaccording to standard protocols, including methanol fixation. Afterincubation with the primary antibody (affinity purified polyclonalrabbit anti-HPV-16 E7 antibody), and secondary antibody (FITC-conjugatedanti-rabbit IgG, Dianova, Hamburg, Germany), cells were washed andembedded in Fluoromount G (Biozol, Eching, Germany). Samples were viewedby indirect immunofluorescence microscopy using the confocal scanningsystem MicroRadiance (Bio-Rad, Munich, Germany) in combination with aZeiss Axiophot microscope. The following filters were used forFITC-derived fluorescence: excitation at 488 nm, emission at 515-530nm). In these experiments, HPV-16 E7 were detected in 5-10% of thetransfected cells, whereas no signal was obtained in cells that havebeen transfected with the empty expression vector. This result clearlyproves that the antibody is specific for the E7 protein and does notdetect any non-specific background under these conditions. The superiorproperties of the antibodies of the invention can be furthermoreillustrated in the following experiment: To calibrate the antibodies,human U2OS cells were transfected with a CMV-driven expression vectorfor HPV-16 E7 and the staining of transfected cells by the antibodies asdescribed herein was compared to the staining pattern obtained withcommercially available antibodies from SantaCruz Biotechnology (ED17) orfrom Zymed Laboratories (8C9X). Staining was analysed by indirectimmunofluorescence and evaluated by confocal microscopy. In theseexperiments, the commercially available antibodies (obtained fromSantaCruz Biotechnology and Zymed Laboratories) were employed inaccordance with manufacturers recommendations and gave high, unspecificbackground staining in all cells. Yet, no specific signal for detectionof E7 antigene in the transfected cells could be obtained with theseprior art antibodies. The corresponding results are documented inappended FIGS. 10 and 11. In contrast, the antibodies according to theinvention are able to specifically detect expressed E7 (positive signalsin 5-10% of the transfected cells) and reveal no signal in cellstransfected by an empty expression vector. These results indicate thatthe antibodies described herein recognize only the E7 protein, whereasthe commercially available antibodies used in the study recognizeunrelated antigens in the preparation. When tested inimmunohistochemical stainings, there was a high and apparentlynon-specific background obtained with the antibodies obtained fromSantaCruz Biotechnology or Zymed Laboratories in tissues derived fromcervix carcinoma patients, as well as in tissue derived from normalcervix. Furthermore, the positive signals obtained with the SantaCruzantibodies were not restricted to the area that is cytologicallyrecognized as tumor tissue, but were also present in the non-tumortissue. In contrast to these results, staining of the biopsy material bythe antibody according to the invention yielded positive results onlyfor HPV-16 positive patients. As can be seen in FIG. 4, staining wasclearly confined to the area of the tumor.

EXAMPLE 8 Immunohistochemical Detection of HPV-16 E7 in Biopsies Derivedfrom Cervical Carcinomas

Immunohistochemistry was performed on paraffin-embedded sections ofHPV16 positive biopsies derived from cervical carcinomas and controltissue specimens. The paraffin-embedded tissue specimens were sectionedat 5 μm. Sections were mounted on slides, deparaffinized in xylol (2×10minutes), incubated for 5 minutes each in 100%, 90%, 80% and 70% ethanoland blocked in 5% H₂O₂ in absolute methanol for 15 minutes. Beforeimmunostaining the sections were washed twice in TRIS buffer (7.75 gTris-HCl pH 7.5, 8.78 g NaCl ad 1 liter aqua dest) and processed for a15 minutes blocking reaction in diluted (1:10 in TRIS buffer/1% BSA)goat-serum (DAKO, Hamburg, Germany). Sections were washed in TRIS/1% BSAbuffer and incubated with the first antibody (affinity purifiedpolyclonal rabbit anti-HPV-16 E7 antibody) for 1 hour at roomtemperature in buffer B (10 μg/ml BSA/10 μg/ml NIH3T3 lysate in PBS).The samples were rinsed twice in TRIS/1% BSA buffer and incubated for 1hour at room temperature with the second antibody (Biotin-conjugatedanti-rabbit IgG, DAKO, Glostrup, Denmark). After the washing step inTRIS/1% BSA, ExtrAvidin-conjugated peroxidase solution (AmershamBiosciences, Vienna, Austria) was added and the samples were incubatedfor 1 hour at room temperature, rinsed in TRIS buffer and processed forstaining. Bound antibodies were visualized with DAB(3.3′-diaminobenzidine) (Sigma, Vienna, Austria) as substrate chromogen.Slides were counterstained with Hemalaun and coverslipped using Eukitt(Merck; Darmstadt, Germany). Brightfield microscopy with photography wasperformed using a Leica DMRB microscope and a Nikon Coolpix 995 camera.

A further protocol comprises the following steps:

Immunohistochemistry was performed on paraffin-embedded sections ofHPV-16 positive biopsies derived from cervical carcinomas and controltissue specimens. The paraffin-embedded tissue specimens were sectionedat 2 and 5 μm. Sections were mounted on slides, deparaffinized in xylol(2×10 minutes), incubated for 5 minutes each in 100%, 90%, 80% and 70%ethanol and blocked in 5% H₂O₂ in absolute methanol for 15 minutes.Before immunostaining the sections were washed twice in TRIS buffer(7.75 g Tris-HCl pH 7.5, 8.78 g NaCl ad liter aqua dest/0.1% Tween 20)and processed for a 15 minutes blocking reaction in diluted (1:10 inTRIS buffer/1% BSA, 0.1% Tween 20) goat serum (DAKO, Hamburg, Germany).Sections were washed in TRIS/1% BSA/0.1% Tween 20 buffer and incubatedwith the first antibody (affinity purified polyclonal rabbit anti-HPV-16E7 antibody) for 1 hour at room temperature in buffer B (10 μg/ml BSA/10μg/ml NIH3T3 Lysate , 0.1% Tween 20 in PBS). The samples were rinsedtwice in TRIS/1% BSA/0.1% Tween 20 buffer and incubated for 1 hour atroom temperature with the second antibody (Biotin-conjugated anti rabbitIgG, DAKO, Glostrup, Denmark). After the washing step in TRIS/1% BSA,ExtrAvidin-conjugated peroxidase solution (Amersham Biosciences, Vienna,Austria) was added and the samples were incubated for 1 hour at roomtemperature, rinsed in TRIS buffer and processed for staining. Boundantibodies were visualized with DAB (3.3′diaminobenzidine) (Sigma,Vienna, Austria) as substrate chromogen. Slides were counterstained withHemalaun and coverslipped using Eukitt (Merck, Darmstadt, Germany).Brightfield microscopy with photography was performed using a Leica DMRBmicroscope and a Nikon Coolpix 995 camera.

EXAMPLE 9 Immunohistochemical Detection of HPV-16 E7 in Prostate DerivedTissue

Biopsies were taken from 60 prostate carcinoma patients and samples from60 patients were applied to an object slide together with negativecontrols. These slides are known to the expert as “tissue microarrays”.Tissue microarrays were stained with antibodies to HPV-16 E7 asdescribed in FIG. 5 for cervical biopsies. In this experiment, a subsetof the carcinoma biopsies stained positive for HPV-16 E7, whereas otherbiopsies from different prostate cancer patients were staining negative.In these experiments, HPV-16 E7 was detected in roughly 10% of theprostate carcinoma specimens analyzed. This result suggests that thesubset of the prostate carcinomas express high levels of HPV-16 E7 andthereby provide evidence for a role of HPV-16 E7 in prostate carcinoma.

EXAMPLE 10 Detection of HPV-16 E7 (Onco-)Protein in Pre-Neoplastic andNeoplastic Cells from Ectocervical Smears (PapSmear)

To determine the presence of HPV-16 E7 protein in ectocervical smears(PapSmear, routinely used for cervical cancer screening), superficialcells were obtained by cervical smear examination (PapSmear) from womenwith normal cervical squamous epithelia (healthy control) and cervicalsquamous intraepithelial lesions. Biopsies were taken at the departmentfor Gynecology and Obstetrics at the University Hospital inInnsbruck/Austria. Biopsies were taken from nine cytologically normalpatients and from twenty patients with abnormal cytological appearance(classified as PapIII by the physician). Superficial cells were obtainedby cervical smear examination (PapSmear) from women with normal cervicalsquamous epithelia (healthy control) and cervical squamousintraepithelial lesions. Cells were streaked out on a glass slide andair dried. Subsequently, cells were fixed in 5% H₂O₂ (freshly dissolvedin absolute methanol) for 15 minutes. Before immunostaining the sectionswere washed twice in TRIS buffer (7.75 g Tris-HCl pH 7.5, 8.78 g NaCl ad1 liter aqua dest) and processed for a 15 minutes blocking reaction indiluted (1:10 in TRIS buffer/1% BSA) goat-serum (DAKO, Hamburg,Germany). Sections were washed in TRIS/1% BSA buffer and incubated withthe first antibody (affinity purified polyclonal rabbit anti-HPV-16 E7antibody described herein) for 1 hour at room temperature in buffer B(10 mg/ml BSA /10 mg/ml NIH3T3 lysate in PBS). The samples were rinsedtwice in TRIS/1% BSA buffer and incubated for 1 hour at room temperaturewith the second antibody (Biotin-conjugated anti-rabbit IgG, DAKO,Glostrup, Denmark). After the washing step in TRIS/1% BSA,ExtrAvidin-conjugated peroxidase solution (Amersham Biosciences, Vienna,Austria) was added and the samples were incubated for 1 hour at roomtemperature, rinsed in TRIS buffer and processed for staining. Boundantibodies were visualized with DAB (3.3′-diaminobenzidine) (Sigma,Vienna, Austria) as substrate chromogen. Slides were counterstained withHemalaun and coverslipped using Eukitt (Merck, Darmstadt, Germany).Brightfield microscopy with photography was performed using a Leica DMRBmicroscope and a Nikon Coolpix 995 camera.

Smears were stained by immunohistochemistry using the affinity-purifiedanti-HPV-16E7 antibody described herein. A representative example isshown in FIG. 6: The antibodies did not stain superficial cells incervical smear from normal HPV-DNA negative ectocervix (FIG. 6A, patientID SM28961), only normal basophile (grey) superficial cells with normalnucleus-cytoplasm relation are visible in this smear. In parallel, aectocervical smear from a patient (patient ID WM20276), that had beenclassified as PapIIID and which was typed as HPV-16 DNA positive by PCRanalysis, was analyzed by immunohistochemistry. A biopsy taken from thepatient one day later revealed CINIII phenotype; laterimmunohistochemical analysis demonstrated high level expression ofHPV-16 E7 in the tumor cells. Only a few normal basophile (grey/blue)superficial cells with normal nucleus-cytoplasm relation can berecognized. However, roughly 50% of the cells show enlargednucleus-cytoplasm relation. These so-called koilocytes are stained bythe E7 antibodies as indicated by the brown colour. Only these cells arestained by the E7 antibodies but not the normal squamous epithelialcells and columnar epithelium cells (usually contained in ectocervicalsmears). This demonstrates that the anti-HPV-16 E7 antibodies describedherein provide a highly specific and sensitive marker for the detectionof abnormal precursor malignant cells in cervical smear preparations(Pap Smears).

EXAMPLE 11 Detection of HPV-16 E7 Protein in Pre-Neoplastic andNeoplastic Cells from Ectocervical Smears

According to Example 10, a further evaluation was carried out underlocal regulations in a blinded trial using patient material obtained atthe University Hospital in Innsbruck/Austria. The evaluation wasperformed by experienced pathologists of the department of Gynecologyand Obstetrics of the University Hospital Innsbruck/Austria who alsovalidated the Pap smears and biopsies, respectively. For screening, Papsmears were taken from women with normal cervical squamous epithelia(healthy control) and cervical squamous intraepithelial lesions. Papsmears were collected with the consent of patients. In case of positivecytology, biopsies were taken at the department of Gynecology andObstetrics at the University Hospital in Innsbruck/Austria.

From 23 women, two smears were taken: one for conservative examination(Papanicolao staining) and one for anti-HPV-16 E7 staining, using theaffinity-purified anti-HPV-16 E7 antibody described herein, followingthe protocol described herein in example 10.

From the 23 Pap smear samples examined, 8 specimens had normalcytological appearance (classified as Pap II or lower, according to theMunich II classification (Soost H J. The Munich nomenclature; RecentResults Cancer Res 1993;133:105-11) and were tested by anti-HPV-16 E7staining, using the affinity-purified anti-HPV-16 E7 antibody/serum ofthe invention.

In 15 specimens with abnormal cytological Pap smear appearance(classified either “higher than Pap II” or “Pap II, unclear”), HPVgenotyping by PCR, conservative histomorphological examination ofcervical tissue biopsy and anti-HPV-16 E7 staining, by using theaffinity-purified anti-HPV-16 E7 antibody, was performed.

Excluding smears that were not assessable because of mucus or few cells,the results (Tab.1) show a clear correlation between an abnormalhistology and the anti-HPV-16 E7 staining. As already demonstrated inExamples 10, it can be shown in the present invention that anti-HPV-16E7 antibodies described herein provide a highly specific and sensitivemarker for the detection of abnormal precursor and malignant cells inPap smears. TABLE 1 Anti-HPV-16 E7 antibodies in Pap smears HPV-TypeAnti-HPV-16 E7 Nr PCR Pap class Histology staining 1 16 rezid. PapIIID/IV CIN III + 2 —*⁾ rezid. Pap IIID CIN I + 3 31 Pap IV CIN III notassessable, mucus 4 33, 58 rezid. Pap IIID CIN I not assessable, mucus 516 Pap II unclear CIN III +/−, few cells 6 —*⁾ Pap II unclear CIN III +7 16 Pap IIID PE Carcinoma + 8 —*⁾ Pap IIID CIN I + 9 —*⁾ rezid. PapIIID CIN I + 10 39 (week) Pap IV CIN III not assessable 11 16 Pap IV CINIII + 12 —*⁾ Pap IIID CIN I + 13 16 Pap III PE Carcinoma few cells 14 56Pap IV CIN III not assessable 15 16 rezid. Pap IIID CIN II + 16 ND PapII ND − 17 ND Pap II ND − 18 ND Pap II ND − 19 ND Pap II ND − 20 ND PapII ND − 21 ND Pap II ND − 22 ND Pap II ND − 23 ND Pap II ND −*⁾no HPV detectableND: not determined

EXAMPLE 12 Detection of HPV-16 E7 in Tissue Homogenates by SandwichELISA

Control of HPV-16 E7 Gene Expression in Biopsies by Western Blot

The expression level of the HPV-16 E7 protein was studied in biopsymaterial derived from HPV-16 DNA positive cervical carcinoma patientsand in histologically normal tissue specimens obtained from patients(HPV-DNA negative by PCR) who underwent hysterectomy for diseasesunrelated to the cervix uteri. Three HPV-16 DNA positive and sevenunrelated cervical biopsies were analysed in Western blot experimentsusing the affinity purified anti-HPV-16 E7 antibodies. The specimensderived from HPV-16 DNA positive cervical carcinoma patients were allpositive, whereas in the unrelated tissues E7 was not detectable (FIG.1). In one biopsy (# 2424) the E7 protein level was as high as in CaSkicells, a cell line derived from a HPV-16 positive cervical carcinoma(Schwarz (1985 Nature 314, 111-119)). In the other HPV-16-positivespecimens the E7 level was lower; however, the different expressionlevels in the individual biopsies can be explained by the fact that theportion of tumor material in a given biopsy differs. No signal wasobtained with cervical cancer biopsies derived from HPV-45 positivepatients.

16E7-ELISA for Detection of HPV-16 E7 in Liquid Samples Derived fromCervical Biopsies

To establish detection of HPV-16 E7 in liquid samples, 96-well ELISAplates were coated with IgG fractions derived from the polyclonalantibody described in the invention. Coated plates were incubated withcrude lysates derived from E. coli-expressing HPV-16 E7 and control E.coli lysates. This experiment was used to determine the effectivethreshold value for reliable detection of 16 E7 antigen. To this end,affinity-purified anti-HPV-16 E7 antibodies were conjugated withhorseradish peroxidase. The conjugate was added to the plate and afterfour washing steps, 3,3′,5,5′-tetra-methylbenzidine (TMB; BoehringerMannheim # 784 974) was added. After incubation for one hour, conversionof TMB was analyzed by densitometric analysis at 450 nm, using a DynatecELISA reader. The values obtained for E. coli lysate were plottedrelative to protein concentration and used to determine the thresholdvalue for the absorption. The background value plus three standarddeviations were used to calculate the threshold value above which asample was considered E7-positive. In the present experiment, thethreshold was set to A₄₅₀>0.16. In a second step, tissue homogenates ofthe human biopsies described above were prepared, diluted in ELISAbuffer and analyzed by 16E7 ELISA, as described above. Results are shownin appended Table 2. TABLE 2 Comparative analysis of HPV-16 E7expression in tissue biopsies by 16E7 ELISA and Western blot Biopsiesfrom five cervical carcinoma patients and biopsies from fivehysterectomy patients without cervical neoplasia (control) were analyzedfor their content of HPV-16 E7 both by ELISA and Western blottechniques. The table gives the absorption obtained in ELISA along withits evaluation (cutoff A₄₅₀ > 0.16) and results obtained by Western blot(see FIG. 1; grading derived from visual inspection). Also indicated isthe HPV DNA status of the patients, as determined by PCR analysis. 16E7-ELISA Biopsy # A₄₅₀ pos/neg 16E7 Western HPV DNA comment 1839 0.08negative − negative control 1867 0.02 negative − negative control 24130.55 positive ++ HPV-16 3358 0.06 negative − negative control 3366 0.09negative − negative control 2227 0.04 negative − HPV-45 2257 0.08negative − HPV-45 2295 0.32 positive + HPV-16 2424 1.4 positive +++HPV-16 2622 0.03 negative − negative control

In the above described example 12, the following methods were employed

1.16 E7-ELISA

Coating

Affinity-purified polyclonal antibodies from rabbits immunized withhighly purified native HPV-16 E7 proteins as described in this inventionwere precipitated by addition of ammonium sulfate to a finalconcentration of 45%, followed by centrifugation. After threeconsecutive precipitations, the antibody was dissolved in water,dialyzed 3× against ice-cold PBS and used at a final concentration of 2μg per ml to coat ELISA plates (Nunc, Vienna).

Conjugation

2 mg of affinity-purified antibodies according to the invention wereconjugated with horseradish peroxidase. Briefly antibodies at 2 mg/ml inPBS (1:10 diluted) were dialyzed overnight at 4 C against sodiumcarbonate buffer (0.01 M NaHCO3Na2CO3, pH 9.3). POD (Sigma cat. # P6782)was dissolved at 8 mg/ml in water and incubated with 1/10 volume of 0.2MNaJO4 for 20 min at RT in the dark. Subsequently, the POD solution wasdialyzed overnight at 4 C against 1 mM sodium acetate/pH 4.4.

For coupling, the POD solution was adjusted to pH 9.3 and immediatelyincubated with the antibody solution. To this end, 650 μl antibodysolution was added to 215 μl POD solution. The mixture was incubated atRT for 2 h under gentle agitation in the dark. To stop the reaction, 43μl of Na(BH4) solution (4 mg/l aqua bidest.) was added and incubationcontinued for 2 h at RT. The conjugate was dialyzed against PBSovernight at 4 C, thiomersal was added to a final concentration of 0.1%.Conjugate was stored at 4 C.

Assay

After addition of TMB, peroxidase reaction and subsequent densitometricanalyses in the ELISA reader were performed as described by themanufacturer.

2. E. coli Lysates

Construction of the Bacterial Expression Vector for HPV-16 E7

The HPV-16 E7 oncogene was amplified from the vector pX-HPV-16 E7(Mannhardt (2000 Mol. Cell. Biol. 20, 6483-6495)) by PCR using Pfu DNApolymerase as Nde1/BamHI fragment. The sequence was inserted into thebacterial expression vector pET3a (Studier and Moffatt (1986 J. Mol.Biol. 189, 113-130)) prepared as NdeI/BamHI fragment generating thebacterial HPV-16 E7 expression vector pET3a-HPV-16 E7. The sequenceencoding for HPV-16 E7 was verified by sequencing.

Preparation of Bacterial Lysates

The expression vector pET3a-HPV-16 E7/Clone 17 was transformed into E.coli strain BL21 (DE3) pLysS. The bacteria were grown to OD₆₀₀=0.5 andthe expression of the E7 protein was induced for 3 hours at 37° C. byadding IPTG (Biomol, Hamburg, Germany) to a final concentration of 0.4mM. For control lysates, the maternal strain E. coli (BL21 (DE3)pLysS)was used.

Bacteria were harvested by centrifugation for 10 minutes at 5 000×g. Thecell pellets were frozen in 20 ml ice-cold lysis buffer (50 mM KCl, 20mM H₂KPO₄ [pH 7.8], 50 mM DTT, 5% glycerol, 1 μg/ml leupeptin, 1 mMPMSF, 1 mM NaF and 10 μg/ml Aprotinin) per liter bacterial culture.Cells were thawed on ice and lysed by sonication with glass beads(Sigma, Vienna, Austria) using a Sonifier 250 (Branson, Geneva,Switzerland). After centrifugation at 70 000×g for 1 hour, thesupernatant was ammonium sulfate precipitated using 30% saturated(NH₄)₂SO₄ solution. The (NH₄)₂SO4 pellet was dissolved in 150 mM Tris/ClpH 7.8, 10 mM NaCl, dialysed against the same buffer and diluted to afinal concentration of 10 mg/ml total protein.

3. Tissue Lysates

For protein extraction, biopsies were extracted in lysis buffer (10 mMTris pH 7.5, 1% Triton X-100, 100 mM NAF, 0.2 mM PMSF). Samples arevortexed and redissolved by 20 strokes with a Branson sonifier on ice,followed by incubation on ice for 5 min. The sample is repeatedly frozenin liquid nitrogen, rethawed, and subsequently incubated on ice foranother 15 minutes, followed by centrifugation for 30 min at 20.000 g.The supernatant is directly used for Western blot analysis or 16E7ELISA.

4. Western Blot (Immunoblot) Analysis

Cell extracts were separated on a 12.5% sodium dodecyl sulfate(SDS)-polyacrylamide gel, and proteins were transferred to a PVDFmembrane (NEN, Boston, USA). The membrane was incubated in blockingbuffer (0.05% Tween 20/5% low fat milk powder in PBS) for 1 hour at roomtemperature, washed in blocking buffer and incubated with the firstantibody (affinity purified polyclonal rabbit anti-HPV-16 E7 antibody)for 1 hour at room temperature. After washing in blocking buffer,PBS/0.05% Tween 20 and PBS/5% low fat milk powder the membrane wasincubated with the second antibody (peroxidase-conjugated anti-rabbitIgG, Promega, Mannheim, Germany) for 45 minutes at room temperature. Themembrane was washed, and the bound antibodies were visualized by usingthe chemiluminescence Western blotting detection system (NEN, Boston,USA).

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1. An anti-HPV-16 E7 antibody obtainable by (a) eliciting an in vivo humoral response against highly purified HPV-16 E7 protein or a fragment thereof in a non-human vertebrate; and (b) affinity-purifying antibodies as obtained in the eliciting-step (a).
 2. The anti-HPV-16 E7 antibody of claim 1, wherein said highly purified HPV-16 E7 protein or a fragment thereof is recombinantly produced.
 3. The anti-HPV-16 E7 antibody of claim 2, wherein said HPV-16 E7 protein or said fragment thereof is expressed in E. coli.
 4. The anti-HPV-16 E7 antibody of claim 1, wherein said highly purified HPV-16 E7 protein or a fragment thereof is purified by a combination of ion exchange chromatography and gel filtration.
 5. The anti-HPV-16 E7 antibody of claim 4, wherein said purification further comprises, prior to ion exchange chromatography and gel filtration, a protein precipitation step.
 6. The anti-HPV-16 E7 antibody of claim 1, wherein said affinity purification of the obtained antibodies is carried out over immobilized HPV-16 E7 protein or a fragment thereof.
 7. The anti-HPV-16 E7 antibody of claim 6, wherein said HPV-16 E7 protein or a fragment thereof is immobilized on PVDF membranes, nitrocellulose, sepharose, agarose, DEAE-cellulose or DEAE.
 8. The anti-HPV-16 E7 antibody of claim 1, wherein said non-human vertebrate is selected from the group consisting of rat, mouse, rabbit, chicken, sheep, horse, goat, pig and donkey; 9.-11. (canceled)
 12. A method for the preparation of a diagnostic composition comprising the step of formulating the anti-HPV-16 E7 antibody of claim 1 with a diagnostically acceptable carrier, diluent, buffer, or storage solution.
 13. The method of claim 12, wherein said diagnostic composition further comprises suitable means for detection.
 14. A diagnostic composition comprising the anti-HPV-16 E7 antibody of claim 1, optionally comprising a diagnostically acceptable carrier, diluent, buffer, or storage solution.
 15. A kit comprising an anti-HPV-16 E7 of claim 1, or a diagnostic composition comprising the anti-HPV-16 E7 antibody with a diagnostically acceptable carrier, diluent, buffer, or storage solution, and optionally a suitable means for detection.
 16. An in vitro method for the detection of a sexually transmittable disease or cancer comprising the steps of a) incubating a biological sample with anti-HPV-16 E7 antibodies of claim 1; and b) measuring and/or detecting specifically-bound anti-HPV-16 E7 antibodies whereby the presence, the absence or the amount of specifically-bound anti-HPV-16 E7 antibodies is indicative for said sexually transmittable disease or cancer.
 17. The in vitro method of claim 16, further comprising a further step (c), whereby in said step (c) the presence, the absence or the amount of specifically-bound anti-HPV-16 E7 antibodies of step (b) is compared to the presence, the absence or the amount of specifically-bound anti-HPV-16 E7 antibodies in a negative or a positive control sample.
 18. (canceled)
 19. The in vitro method of claim 16, wherein said sexually transmitted disease is an HPV16-infection or wherein said cancer is cervical cancer, breast cancer/mamma cancer, prostate cancer, head and neck cancer, penil cancer and/or anogenital cancer/neoplasia (AIN).
 20. A method for the production of an anti-HPV-16 E7 antibody comprising the steps of (a) eliciting an in vivo humoral response against highly purified, HPV-16 E7 protein or a fragment thereof in a non-human vertebrate; and (b) affinity-purifying antibodies as obtained in the eliciting-step (a).
 21. The method of claim 20, wherein said highly purified HPV-16 E7 protein or said fragment thereof is a native, highly purified HPV-16 E7 protein or a fragment thereof.
 22. The anti-HPV-16 E7 antibody of claim 1, wherein said highly purified HPV-16 E7 protein or said fragment thereof is a native, highly purified HPV-16 E7 protein or a fragment thereof.
 23. The method of claim 16, wherein said biological sample is obtained from Pap-smears, cervical (carcinoma) biopsies, anogenital biopsies, mamma biopsies, head- or neck biopsies or prostate biopsies.
 24. The method of claim 16, wherein said detection is used for evaluating the risk of acquiring a sexually transmitted disease or cancer, for measuring the status of an existing sexually transmitted disease or cancer, or for screening therapy efficiency in the treatment of a sexually transmitted disease or cancer. 